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S.L. Lim et al.
a panel of 13 human MM cell lines (KMS11, MM1R, KMS12BM, H929, KMS18, 8226 LR5, MM1S, KMS11 res, U266, 8226, KMS28BM, 8226 P100V, MM1S res) using an in vitro proliferation assay (MTT, 72 h). Cell lines included melphalan resistant (8226 LR5), steroid resistant (MM1R), bortezomib resistant (8226 P100V), and lenalidomide resistant (KMS11 res and MM1S res) cell lines. Their cyto- genetics varied; some were associated with a poor prog- nosis [e.g. t(4:14): KMS11, KMS28BM, H929; t(14:16): MM1S, 8226]. ARV 825 was more potent than either OTX 015 or pomalidomide alone against both KMS11 (IC50 for ARV 825, OTX 015 and pomalidomide: 9 nM, 130 nM, >1000 nM, respectively) and KMS28BM cells (IC50 for ARV 825, OTX 015 and pomalidomide: 137 nM, 240 nM, >1000nM, respectively) (Figure 1B). All MM cell lines were sensitive to ARV 825 with an IC50 ranging from 8 nM to 500 nM except for MM1S res cells (>1000 nM) (Figure 1C). MM1S res cells (resistant to lenalidomide) had significantly reduced CRBN levels (Online Supplementary Figure S1A and B). The MM1S res cells had a 40-fold reduc- tion in expression of CRBN compared to parental MM1S cell line due to deletion of one allele of the CRBN gene and a point mutation on the second allele.14 Therefore, because of lack of wild-type CRBN, they had loss of wild-type CRBN expression (western blot, Online Supplementary Figure S1B) associated with a resistance to ARV 825. Likewise, KMS11 res cells (resistant to lenalidomide) also have a structural deletion of CRBN with reduced CRBN expression15 (Online Supplementary Figure S1A and B) and were 8.3-fold more resistant to ARV 825 compared to its parental cells. Overexpression of wild-type CRBN in MM1S res cells rescued this resistant cell line and increased its sensitivity to ARV 825 (IC50=800 nM) (Online Supplementary Figure S1C). KMS11 cells are the most sensitive, with an IC50 of 8.5 nM; in contrast, KMS28BM is a relatively more resistant cell line (IC50=137 nM) (Online Supplementary Table S1). ARV 825 decreased clonogenic growth of KMS11 and KMS28BM in a dose-dependent manner (Figure 1D).
The other PROTAC (MZ1) significantly suppressed growth of the lenalidomide resistant cells (MM1S res and KMS11 res) as well as some of the other MM cells. However, KMS18, U266, 8226, 8226 LR5 and 8226 P100V were relatively resistant to MZ1 (Figure 1E). IC50s are shown in Online Supplementary Table S2.
The bortezomib-resistant cell line (8226 P100V) is also relatively resistant to ARV 825 (IC50=500 nM). Consistently, the BRD 4 degradation in 8226 P100V cells after treatment with different doses of ARV 825 (50 nM, 100 nM, 200 nM) only modestly decreased when com- pared to its parental strain (8226). 8226 P100V has up-reg- ulated mRNA expression of multidrug resistance-associat- ed protein 1 (MRP-1, encoded by the ABCC1 gene) com- pared to its parental strain (8226) (Online Supplementary Figure S1D).
ARV 825 inhibit growth of primary myeloma cell samples
The effect of ARV 825 (10 nM to 300 nM) on three MM patient samples was evaluated. The PROTAC significantly inhibited growth of these MM patient samples which include one multiple relapsed patient (Patient 3). 10 nM of ARV 825 inhibited the growth of MM Patients 1, 2 and 3 by 82%, 93%, and 64%, respectively (Figure 1F).
A
B
Figure 4. ARV 825 induced cell cycle arrest and apoptosis of multiple myeloma (MM) cells. (A) Cell cycle: KMS11 and KMS28BM MM cells were treated for 48 hours (h) with either ARV 825 (2.5-10 nM or 25-50 nM, respectively, 48 h) or diluent control [dimethyl sulfoxide (DMSO)], stained with propidium iodide (PI) and analyzed by flow cytometry. Histograms showed proportion of cells in differ- ent phases of cell cycle. Representative of three independent experiments. (B) Apoptosis: KMS11 and KMS28BM cells treated with of ARV 825 (10-40 nM or 100-400 nM, respectively, for 48 h), stained with annexin V-FITC and PI, and ana- lyzed by flow cytometry. Histograms represent percentage of apoptotic cells. Mean±Standard Deviation of three independent experiments. *P≤0.01; **P≤0.001; ***P≤0.0001 for ARV 825 versus control.
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haematologica | 2019; 104(6)