Leaders in Pharmaceutical Business Intelligence Group, LLC, Doing Business As LPBI Group, Newton, MA

Fig. 2. Chronic exposure to degraders induces MDR1 expression and drug efflux activity.

(A) ABCB1 mRNA levels in parental and degrader-resistant cell lines as determined by qRT-PCR. Data are means ± SD of three independent experiments. ***P ≤ 0.001 by Student’s t test.

(B) Immunoblot analysis of MDR1 protein levels in parental and degrader-resistant cell lines. Blots are representative of three independent experiments. (C to E) Immunofluorescence (“IF”) microscopy of MDR1 protein levels in A1847 dBET6-R

(C), SUM159 MZ1-R

(D), and Thal-R A1847 cells

(E) relative to parental cells. Nuclear staining by DAPI. Images are representative of three independent experiments. Scale bars, 100 μm.

(F) Drug efflux activity in A1847 dBET6-R, SUM159 MZ1-R, and Thal-R A1847 cells relative to parental cells (Par.) using rhodamine 123 efflux assays. Bars are means ± SD of three independent experiments. ***P ≤ 0.001 by Student’s t test.

(G) Intracellular dBET6 levels in parental or dBET-R A1847 cells transfected with a CRBN sensor and treated with increasing concentrations of dBET6. Intracellular dBET6 levels measured using the CRBN NanoBRET target engagement assay. Data were analyzed as % of DMSO control, presented as means ± SD of three independent assays. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 by Student’s t test. (H and I) FISH analysis of representative drug-sensitive parental and drug-resistant A1847

(H) and SUM159

(I) cells using ABCB1 and control XCE 7 centromere probes. Images of interphase nuclei were captured with a Metasystems Metafer microscope workstation, and the raw images were extracted and processed to depict ABCB1 signals in magenta, centromere 7 signals in cyan, and DAPI-stained nuclei in blue. (J and K) CpG methylation status of the ABCB1 downstream promoter (coordinates: chr7.87,600,166-87,601,336) by bisulfite amplicon sequencing in parent and degrader-resistant A1847

(J) and SUM159

(K) cells. Images depict the averaged percentage of methylation for each region of the promoter, where methylation status is depicted by color as follows: red, methylated; blue, unmethylated. Schematic of the ABCB1 gene with the location of individual CpG sites is shown. Graphs are representative of three independent experiments. (L and M) Immunoblot analysis of MDR1 protein levels after short-term exposure [for hours (h) or days (d) as indicated] to BET protein degraders dBET6 or MZ1 (100 nM) in A1847

(L) and SUM159

(M) cells, respectively. Blots are representative of three independent experiments. (N to P) Immunoblot analysis of MDR1 protein levels in A1847 and SUM159 cells after long-term exposure (7 to 30 days) to BET protein degraders dBET6

(N), Thal SNS 032

(O), or MZ1

(P), each at 500 nM. Blots are representative of three independent experiments. (Q and R) Immunoblot analysis of MDR1 protein levels in degrader-resistant A1847

(Q) and SUM159

(R) cells after PROTAC removal for 2 or 7 days. Blots are representative of three independent experiments. Related data are in fig. S2 and data file S2.