Supplemental Information. Inhibition of the Proteasome b2 Site Sensitizes. Triple-Negative Breast Cancer Cells

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1 Cell Chemical Biology, Volume 24 Supplemental Information Inhibition of the Proteasome b2 Site Sensitizes Triple-Negative Breast Cancer Cells to b5 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, and Alexei F. Kisselev

2 a b μm LU-102 MDA-MB μm LU-102 c HCC μm LU μm NC-021 d Cathepsins e CombinaCon indexes, Cfz3uM LU-102 f NC-021 (5μM) LU-102 (3μM) Cfz (150nM) g Viable cells, % ctl Figure S1.

3 a ABPs parental T Cfz (nm) i /5i /5i i b /i c wt T1A e d cell culture parental ΔT1 vehicle tumors ΔT1 tumors Cfz tumors and i i V I a b l e c e l l s (% c o n t r o l) I3 wt T1 i parental T1A vehicle tumors T1A tumors Cfz tumors i i f cell lines cell lines cell lines Figure S2.

4 a, Cfz, CfzNC-021 CfzLU-102 b SUM149, Cfz SUM149, CfzNC-021 SUM149, CfzLU-102 Figure S3.

5 Santa Cruz ancbody Cell signalling ancbody accn gly Nrf1 FL Nrf1 cl Nrf1 accn gly Nrf1 FL Nrf1 cl Nrf1 Figure S4.

6 Supplemental Figure Legends Figure S1 (related to Figure 2). NC-021 and LU-102 were used at site-specific concentrations that did not inhibit cathepsins or cell growth. a) NC-021 is a more potent analogue of NC-001, the inhibitor we developed previously (Britton et al., 2009). The Proteasome-Glo assay was used to compare active site inhibition by NC-001 and NC-021. b) 5 µm NC-021 and 3 µm LU-102 are site-specific in multiple cell lines. Cells were treated with inhibitors for 1h, and proteasome inhibition was determined with active site probes. c) Cells were treated with a 1h pulse of inhibitor every 24h, and cell number was determined via automated cell counter. d) Cells were treated for 6h with LU-102 and cumulative cathepsin B,H,L, and S activity was measured in whole cell extract with Z-FR-amc at ph 6 (Britton et al., 2009; Mirabella et al., 2011). e) Combination indexes (CIs) for experiment in Figs. 2 d,e were calculated using Calcusyn software. f) cells were treated for 1h. Cells were stained with Annexin V after 24h and analyzed by flow cytometry. g) cells were either co-treated with Btz and LU-102 for 1h, or treated with Btz for 1h, and then treated with LU-102 for 47h. Viable cells were assayed with Alamar Blue 48h after start of the treatment. Figure S2 (related to Figure 3). Characterization of CRISPR cell lines. a) ß5/5i cross-reacts with BODIPY-NC ΔT1 cells were treated with the indicated concentration of Cfz for 1h, harvested, and proteasome activity was determined with activity-based probes. The disappearance of the faint band in the position with Cfz treatment indicates that this band is attributed to. b) Activity of each active site was measured with activity-based probes. and i are graphed on the same axis to show relative contribution. i was too weak to quantify accurately. c) Inactivation of the active sites targeted by LU-102 and NC-021 eliminates their sensitizing effects. Cells were treated with Cfz, 3 µm LU-102, 5 µm NC-021 for 1h. After 48 hours viable cells were measured with Alamar Blue. d) Cells were treated for 72h with doxorubicin and viable cells were determined with Alamar Blue. e) ΔT1 and T1A tumors did not revert to wild-type or after Cfz treatment. Tumors were harvested at the end of experiment in Fig. 3e and proteasome activity was determined with active site probes. f) Growth rate of mutant clones. Cell number was determined with an automated cell counter. Figure S3 (related to Figure 4). LU-102 blocks proteasome activity recovery in Cfz-treated cells. a,b) Cells were treated for 1h with 100nM Cfz, plus 5µM NC-021 or 3µM LU-102 and harvested at indicated times. Occupancy was determined with activity-based probes. Data are mean ± S.E.M of 2-3 biological replicates. Figure S4 (related to Figure 5). CfzLU-102 treatment causes accumulation of the full-length nonglycosylated form of Nrf1 in cells. Cells were treated 2h with 10uM NMS-873 and harvested immediately, or treated 1h with vehicle or 100nM Cfz and 3uM LU-102 and harvested at 16h. Cells were analyzed by western blot with the two different Nrf1 antibodies.

7 Supplemental Tables Table S1 (related to Fig 3). Numbers of mice per group in the experiment in Fig. 3e. Treatment Vehicle Cfz mutant Number of mice wt 10 8 T1A wt ΔT1 8 8

8 Table S2 (related to Fig. 6). LU-102 sensitizes cell lines of other cancer types to Cfz and Btz. Treatment Cfz CfzLU-102 Btz BtzLU-102 IC 50 (C.I., nm) Non-small cell lung cancer H (79-242) 1 (1-2) 112 (49-134) 13 (9-18) Hop (82-177) 25 (18-35) 162 ( ) 30 (26-36) H ( ) A ( ) Renal cancer TK (76-131) Ovarian cancer OVCAR4 398 ( ) (9-25) 2 (n.d.) 6 (n.d.) 32 (21-48) ( ) 398 ( ) 35 (n.d.) 316 ( ) (24-40) 56 (38-81) 19 (11-37) 36 (28-48) Cells were treated as in Table 1, except that LU-102 treatment was done as a 47h chase instead of a co-treatment. As LU-102 is an irreversible inhibitor, co-treatment and pulse treatment are equivalent (Fig. S1g).

9 Supplemental Experimental Procedures Cell culture media All cells were grown at 37 C in 5% CO 2, in media listed below. All media were additionally supplemented with penicillin, streptomycin and plasmocin. Cell Line Base media Supplements SUM149 DMEM/F12 50:50 5% FBS, 1ug/mL hydorcortisone, 4.8ug/mL human recombinant insulin, 10mM HEPES ph 7.3, 4mM L-glutamine and genetically engineered derivatives DMEM/F12 50:50 5% FBS MDA-MB-468 DMEM 10% FBS 293-FT DMEM 10% FBS, 0.1mM MEM nonessential amino acids, 6mM L- glutamine, 1mM sodium pyruvate All other cell lines RPMI % FBS NC-021 analytical data 1 H NMR (600 MHz, CDCl 3 ): (br s, 0.5H), (br d, 0.5H, J = 7.2 Hz), (br d, 0.5H, J = 7.2 Hz), (br s, 0.5H), (m, 1H), (m, 1H), (m, 1H), (m, 1H), (m, 6H), (m, 1H), (m, 1H), (m, 3H), 3.31 and 3.29 (two d, 1H, J = 4.8 Hz), 2.90 and 2.87 (two d, 1H, J = 4.8 Hz), (m, 0.5H), (m, 3.5H), (m, 2H), (m, 2H), 1.54 (s, 1.5H), 1.46 (s, 1.5H), (m, 0.5H), (m, 4.5H), (m, 9H). MS (ESI, m/z): calculated for C 26 H 44 N 4 O 6 [MH ] found Immunoblotting and antibodies used Samples were heated for 10 minutes with LDS buffer (Novex NP0007) at 72 o C, fractionated on Bis-Tris gels (Genscript) using MOPS electrode buffer, and transferred to 0.2 µm PVDF membrane (Immobilon, ISEQ00010). Blots were blocked with Odyssey Blocking Buffer (LI-COR, ). The following primary antibodies were used: K48 polyubiquitin (Cell Signalling, 8081); PARP (Cell Signalling, 9542); (Enzo, 8140); (Enzo, 9300); (Enzo, 8895); histone H3 (Bethyl, A A); ORC2 (Cell Signalling, 4736S); and actin (Abcam, 3280). A Santa Cruz anti-nrf1 antibody (13031 (Sha and Goldberg, 2014)) was used for all western blots except for Figs. 5d,h, in which a Cell Signalling anti-nrf1 antibody was used (8052S (Sha and Goldberg, 2016)). Fluorescent-labeled secondary antibodies were used for all primaries except for Cell Signalling anti-nrf1, for which HRP-linked secondary antibodies were used. Fluorescent-labeled secondary antibodies were obtained from LI-COR: goat anti-mouse ( ) and goat anti-rabbit ( ). HRP-linked antibodies were obtained from Cell Signalling (anti-rabbit, 7074P2) and Novex (anti-mouse, A16072). Fluorescent-labeled secondary antibodies were detected with the Odyssey imager (LI-COR) and quantified with the Odyssey software. HRP-linked secondary antibodies were detected with Pierce SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher, 34095).