Volume 24, Issue 2, Pages 218-230 (February 2017) Inhibition of the Proteasome β2 Site Sensitizes Triple-Negative Breast Cancer Cells to β5 Inhibitors and Suppresses Nrf1 Activation  Emily S. Weyburne, Owen M. Wilkins, Zhe Sha, David A. Williams, Alexandre A. Pletnev, Gerjan de Bruin, Hermann S. Overkleeft, Alfred L. Goldberg, Michael D. Cole, Alexei F. Kisselev  Cell Chemical Biology  Volume 24, Issue 2, Pages 218-230 (February 2017) DOI: 10.1016/j.chembiol.2016.12.016 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 218-230DOI: (10. 1016/j. chembiol. 2016 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 The Sensitivity of TNBC Cells to Btz and Cfz Does Not Correlate with Inhibition of the β5 Site (A) TNBC and MM cell lines were treated with Btz for 1 hr, recovered in drug-free medium for 48 hr, and assayed for viable cells with Alamar blue. Dose-response curves were generated by plotting the averages of two to five biological replicates and used to determine the average IC50. See Table 1 for confidence intervals. MM data are from Shabaneh et al. (2013). TNBC subtype assignments are from Charafe-Jauffret et al. (2009). (B and C) Cells were treated with Btz for 1 hr, then immediately assayed for proteasome inhibition using site-specific substrates from Proteasome-Glo. In a parallel experiment, viable cells were quantified 48 hr after treatment as in (A). (D and E) Viable cells were plotted against inhibition of the β5 and β1 sites after Btz treatment. (F and G) Cells were treated with Cfz for 1 hr and analyzed as in (B). (H) Viable TNBC cells were plotted against inhibition of the β5 site after Cfz treatment. Values in (B) to (H) are mean ± SEM of two to three biological replicates. Cell Chemical Biology 2017 24, 218-230DOI: (10.1016/j.chembiol.2016.12.016) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Inhibition of the β2 Subunit Sensitizes Cells to β5 Inhibitors Better than Inhibition of the β1 Subunit (A and B) SUM149 cells were treated for 1 hr with LU-102 (A) or NC-021 (B). The active-site probes MV-151 and BODIPY-NC-001 (Verdoes et al., 2010) were used to determine site inhibition in cell extracts. Band intensities were quantified by densitometry. The graphs are averages ± SEM of two independent experiments. (C) SUM149 cells were treated with NC-021 or LU-102 for 1 hr and viable cells were determined with Alamar blue after 48 hr. (D and E) Cells were treated with Cfz, Cfz + 5μM NC-021, or Cfz + 3 μM LU-102 for 1 hr and viable cells were determined after 48 hr. Combination indexes are listed in Figure S1E. (F) SUM149 cells were treated with 100 nM Cfz, 3 μM LU-102, and 5 μM NC-021 for 1 hr. Cells were stained with annexin V after 36 hr. Values in (C–F) are averages ± SEM of two to three biological replicates. (G) Average IC50s were plotted relative to the average IC50 for Btz or Cfz only. (H and I) Viable cells after 1 hr of co-treatment with Cfz + 3 μM LU-102 (H) or Cfz + 5 μM NC-021 (I) were plotted against inhibition of β5 sites by Cfz (as in Figure 1H). NC-021 and LU-102 do not alter inhibition of the β5 sites by Cfz (not shown). Values are averages ± SEM of two to three independent experiments. Cell Chemical Biology 2017 24, 218-230DOI: (10.1016/j.chembiol.2016.12.016) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Mutant CRISPR Cell Lines with Inactive β2 Subunits Are More Effectively Sensitized to β5 Inhibition Than Cells with Inactive β1 Subunits (A) Activity of each active site was determined with activity-based probes. “i” designates subunits of lymphoid-tissue specific immunoproteasomes. The faint band between β1 and β1i in the β1ΔT1 mutant is due to BODIPY-NC-001 cross-reacting with the β5/5i subunit (see Figure S2A). WT, wild-type. (B) Sequences of mutant alleles. T1 is the active-site threonine. Lowercase letters indicate CRISPR-introduced T1A and synonymous mutations. subs., substitution; del., deletion. (C) Western blot confirming expression of mutant subunits. Data are presented as averages ± SEM of two independent experiments. (D) Cells were treated with Cfz for 1 hr and viable cells were determined at 48 hr. Data are averages ± SEM of three to seven biological replicates. (E) Cells were injected into the mammary fat pads of female NSG mice. Day 0 indicates the day that tumors were deemed palpable, defined as 14 mm3. Mice were treated intravenously with 1.5 mg/kg Cfz twice weekly on consecutive days. ***p < 0.001, two-way ANOVA, compared with vehicle control, n = 8–14 per group (Table S1). Cell Chemical Biology 2017 24, 218-230DOI: (10.1016/j.chembiol.2016.12.016) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 β2 Inhibition Increases Accumulation of Ubiquitinated Protein and Inhibits Proteasome Activity Recovery in Cfz-Treated Cells (A and C) Cells were treated for 1 hr with 100 nM Cfz, 3 μM LU-102, and 5 μM NC-021, harvested at the times indicated, and analyzed by western blot. (B) Cells were treated as in (A) and harvested. Caspase activity was determined in cell extracts using the caspase substrate Ac-DEVD-amc. (D) CRISPR cell lines were treated for 1 hr with 100 nM Cfz and harvested at 9 hr, then analyzed by western blot. (E) Cells were treated for 1 hr with the same concentrations of inhibitors as in (A) and (B), and β5 occupancy was determined with activity-based probes as in Figures 2A and 2B. See Figure S3 for β1 and β2 occupancies. Data in (B–E) are presented as means ± SEM of two to three biological replicates. Cell Chemical Biology 2017 24, 218-230DOI: (10.1016/j.chembiol.2016.12.016) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Co-inhibition of β5 and β2 Suppresses Soluble, Active Nrf1 and Prevents Upregulation of Proteasome Genes (A–C) Cells were treated for 1 hr with 100 nM Cfz, 3 μM LU-102, and/or 5 μM NC-021 (A and B), or 100 nM Cfz (C), harvested at times indicated, lysed with CHAPS buffer, and analyzed by western blot. Cfz + NC-021 at 18 hr is loaded on both gels to allow comparison. Representative results of two experiments are shown. FL, full-length; cl, cleaved. (D) SUM149 cells were treated for 1 hr and harvested at 16 hr. The soluble fraction was isolated with CHAPS buffer, the pellet was treated with DNase and high-salt to isolate the chromatin-bound fraction, and aggregates were subsequently solubilized by sonication in 2% SDS. Fractions were analyzed by western blot. Origin Recognition Complex subunit 2 (ORC2) is a marker for chromatin-bound proteins. (E) MDA-MB-231 cells were treated as in (A), harvested at 10 hr, and mRNA was measured by RT-PCR. mRNA levels are relative to the control gene PGK1. (F) Cells were treated as in (C), harvested at 12 hr, and mRNA measured by RT-PCR. (G and H) Cells were treated with 60 nM control, Nrf1, or DDI2 siRNA, then treated with indicated compounds for 1 hr. Samples for western blot were harvested at 16 hr. Remaining cells were stained with annexin V after 36 hr. (I) Model for the effect of proteasome inhibition on Nrf1 regulation. ***p < 0.001, **p < 0.01, *p < 0.05, unpaired t test; ns, not significant. Data in (E) to (H) are presented as mean ± SEM of two to five biological replicates. Cell Chemical Biology 2017 24, 218-230DOI: (10.1016/j.chembiol.2016.12.016) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 LU-102 Sensitizes Cell Lines Derived from Other Solid Tumors to Cfz and Btz (A and B) Percentages of viable cells were plotted against inhibition of the β5 or β1 sites after 1 hr of treatment with Cfz. Cells lines are derived from lung (A549, H23, Hop-62, H226), renal (TK-10), or ovarian (OVCAR4) cancer. The values are averages ± SEM of two to three independent experiments. (C) Average IC50s were plotted relative to the average IC50 for Btz or Cfz only. (D) Percentage of viable cells was plotted against inhibition of the β5 site after 1 hr treatment with Cfz + 3 μM LU-102. LU-102 did not reduce viability when used as a single agent and did not alter inhibition of the β5 site by Cfz (not shown). Data are presented as averages ± SEM of two to three independent experiments. Cell Chemical Biology 2017 24, 218-230DOI: (10.1016/j.chembiol.2016.12.016) Copyright © 2017 Elsevier Ltd Terms and Conditions