1. Volume 26, Issue 11, Pages 3061-3075.e6 (March 2019)
A Pan-ALDH1A Inhibitor Induces Necroptosis in Ovarian Cancer Stem-like Cells 
Ilana Chefetz, Edward Grimley, Kun Yang, Linda Hong, Ekaterina V. Vinogradova, Radu Suciu, Ilya Kovalenko, David Karnak, Cynthia A. Morgan, Mikhail Chtcherbinine, Cameron Buchman, Brandt Huddle, Scott Barraza, Meredith Morgan, Kara A. Bernstein, Euisik Yoon, David B. Lombard, Andrea Bild, Geeta Mehta, Iris Romero, Chun-Yi Chiang, Charles Landen, Benjamin Cravatt, Thomas D. Hurley, Scott D. Larsen, Ronald J. Buckanovich 
Cell Reports 
Volume 26, Issue 11, Pages e6 (March 2019)
DOI: /j.celrep
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2. Cell Reports 2019 26, 3061-3075.e6DOI: (10.1016/j.celrep.2019.02.032)
Copyright © 2019 The Author(s) Terms and Conditions

3. Figure 1 ALDH1A Family Members in Ovarian Cancer
(A) qRT-PCR (i) and in silico (ii) CCLE analysis of ALDH gene expression in various ovarian cancer cell lines.
(B) Analysis of ALDH1A family member DNA deletion and amplification or mRNA expression changes in the ovarian cancer TCGA database.
(C) (i) qRT-PCR confirmation of ALDH1A family member mRNA downregulation with siRNA treatment in PEO4 cells. (ii) Cell counts in the indicated cell lines following ALDH family member downregulation.
(D) Cell viability in FACS sorted CD133+ and CD133− from A2780 and Ovsaho 72 h after ALDH1A1 or ALDH1A3 downregulation.
Error bars represent SDs. Results are a summary of n = 3 independent experiments with at least three technical replicates. Data are presented as mean ± SD with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p <
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4. Figure 2 Identification of an ALDH1Ai, 673A
(A) Effect of the indicated ALDH inhibitors on the percentage of viable CD133+ A2780 cells (absolute CD133+ cells in each group are normalized to untreated controls) by FACS 72 h after treatment.
(B) Quantification of in vitro enzymatic inhibition of ALDH family members.
(C) ALDEFLUOR assay of ALDH activity in live PEO1 cells after treatments with 25 μM DEAB or 673A at the indicated times.
(D) Viability of OVCAR8 cell controls or OVCAR8 siALDH1A3 knockdown cells with and without 673 (i), and 673 dose-response curve for the indicated cell lines comparing transfected controls (ctrl) versus either CRISPR knockdown of ALDH1A1 or ALDH1A3 (ii).
(E) Cell viability of FACS-sorted CD133+/− (A2780, Ovsaho, and OVCAR5) cells 72 h after treatment with 12.5 μM 673A.
(F) Quantification (i) of cell death of single CD133+/ALDH+ (A2780) cells (ii) on microfluidic chips 72 h after treatment.
Error bars represent SDs; n = 3 independent experiments with at least triplicate assays. Data are presented as mean ± SD with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005.
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5. Figure 3 673A Triggers Necroptosis in Ovarian CSCs
(A) (i) FACS analysis of annexin-V and PI stain as indicators of apoptosis in FACS-sorted CD133+ (A2780) cells after treatment with 12.5 μM 673A, 1 μg/ml cisplatin, or 12.5 μM shikonin. (ii) Cell counts of FACS-sorted CD133+ A2780 cells after pretreatment with pan-caspase inhibitor (CI; 20 μM) and caspase 3 inhibitor (C3I; 20 μM) plus 72-h treatment with the indicated concentrations of 673A. (iii) FACS analysis of CD133+ expression after treatments with CI (20 μM) and 673A (12.5 μM).
(B) Immunofluorescence (IF) DAPI nuclear stain (i) and TEM images (5,800×) (ii) of FACS-sorted CD133+ A2780 cells 24 h after treatment with 673A (12.5 μM) (red arrows, mitochondria; blue, rupture of plasma membrane).
(C) IF of HMBG1 localization 24 h after 673A treatment; DAPI was used for nuclear stain.
(D) Intracellular calcium level, using Fluo-4 FACS assay after 673A treatments (i). Cell counts (ii) and CD133 FACS analysis (iii) of A2780 cells after pretreatments with 20 μM BAPTA and treatments with 673A.
(E) FACS of annexin-V-PE/DAPI in PEO4 cells after ALDH1A family member downregulation using siRNA (48 h).
Error bars represent SDs n = 3 independent experiments with at least triplicate assays. Data are presented as mean ± SD with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005.
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6. Figure 4 Role of RIPKs in ALDH Inhibition-Induced Cell Death
(A) Cell counts following 72 hr transfection of RIPK1 siRNA with and without 12.5-μM 673A treatment (i). Cell counts of FACS-sorted CD133+ A2780 (ii) and OVCAR8 (iii) cells after pretreatment with RIPK1 inhibitor Nec-1 and treatments with or without 673A.
(B) Cell counts after 72 h transfection of RIPK3 or control siRNA with or without 12.5 μM of 673A (i). Cell counts of FACS-sorted CD133+ A2780 cells 72 h after pretreatment with 5 μM of MLKL inhibitor necrosulfonamide (NSA) plus treatment with the indicated doses of 673A (ii).
(C) A western blot of PGAM5 protein immunoprecipitated (IP) with FADD, DRP1, and RIPK3 antibodies with or without 673A treatment in CD133+ (A2780).
(D) A western blot analysis of pDRP1, DRP1, and PGAM5 after 673A treatments. Note that controls were on the same blot but were separated, thus the gel break. Exposures for controls and the remaining gel are identical.
(E) A western blot analysis of DRP1 protein in mitochondrial and cytoplasmic fractions (i) and MLKL protein in membranous and cytoplasmic cellular fractions (ii) after 12.5-μM 673A treatment.
Error bars represent SDs n = 3 independent experiments with at least triplicate assays. Data are presented as mean ± SD with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p <
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7. Figure 5
673A Induces Expression of the Mitochondrial Uncoupling Proteins
(A) Count of viable cells of CD133+ and CD133− PEO4 cells after 36-h treatment with transcription inhibitor flavopiridol ± 673A.
(B) qRT-PCR analysis of uncoupling protein 1 (UCP1) and UCP3 mRNA expression in total PEO4 cells (i) and FACS-sorted CD133+/− PEO4 cells (ii) after treatments with 12.5 μM of 673A.
(C) qRT-PCR analysis of UCP1/3 expression 48 h after siRNA downregulation of ALDH1A family members in PEO4 cells.
(D) Cell counts (i) and FACS analysis (ii) of annexin-V/DAPI after 48 h of UCP1 and UCP3 overexpression in FACS-sorted CD133+ A2780 cells.
(E) Cell counts of cells transfected with UCP1 and UCP3 siRNA and treated with 12.5 μM 673A (at 72 h).
(F) Quantification of basal and spare respiratory capacity (i) and ATP production in primary HGSC cells (ii) after treatment with 12.5 μM 673A as assessed by Seahorse.
Error bars represent SDs n = 3 independent experiments with at least triplicate assays. Data are presented as mean ± SD with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005.
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8. Figure 6 The Effect of ALDH Inhibition on Tumor Stemness
(A) (i) Cell counts of SKOv3 cells over time after treatment with 1 μg/mL cisplatin alone or in combination with ALDH inhibitors. (ii) Synergy factor calculations upon treatment of primary ovarian high grade serous (HGS) samples in spheres (Pt152, Pt259, and Pt224) and breast cancer cell lines (HCC3153, HCC1937, HCC1395, and HCC1569).
(B) Surviving fraction after radiation of FACS-isolated CD133+/− A2780 (i) and ALDH+/− SKOv3 (ii) cells after DMSO or 673A treatment and subsequent radiation, and table of radiation-enhancement ratios (iii).
(C) Sphere formation capacity in primary HGS cells with 673A.
(D) Tumor growth (i) and tumor initiation capacity (ii) of 5,000 FACS-sorted viable (annexin-V−/PI−) cells after single-agent and combined therapy with cisplatin and ALDH inhibitors. Number of established tumors was evaluated 3 months following cell injection.
Error bars represent SDs; n = 3 independent experiments with at least triplicate assays. Data are presented as mean ± SD with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.005.
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9. Figure 7 673A Efficacy In Vivo
(A) Tumor volumes for HEY-1, Ovsaho, and A2780. Error bars represent means ± SD; n = 10 mice.
(B) (i) H&E and Ki67 images of Ovsaho tumors. (ii) Quantification of Ki67. Scale bars represent 200 μm for H&E and 100 μm for Ki67.
(C) Volumes for OVCAR8 tumors. Error bars represent means ± SD; n = 10 mice; p < 0.01.
(D) qRT-PCR analysis of UCP1 and UCP3 expression in OVCAR8 tumors; n = 3 mice.
(E) Percentage of mice that were tumor free 6 months after inoculation with 1 × 105 CAOV3 cells and treatment with 21 days of 673A (20 mg/kg for 21 days), cisplatin (1 mg/kg once a week for 3 weeks), or both. Fraction surviving is indicated in the parentheses.
(F) Survival studies during 6 months after i.p. inoculation of 1 × 104 FACS-sorted ALDH+ SKOv3 cells.
(G) Percentage of mice that were tumor free after 9 weeks of treatments in an LSL-K-rasG12D/+PtenloxP/loxP endometrioid ovarian cancer model.
(H) Growth curves of platinum-resistant PDX; n = 5 mice/group for two independent PDX models. The 673A was given daily for 21 days at 20 mg/kg; cisplatin or carboplatin were given weekly.
(I) (i) Combined waterfall plot from four biological replicates (mean R values) of ALDH1A inhibition in SKOv3 tumors in vivo. (ii) Representative of inhibition of ALDH1a1_C303 in two data sets in SKOv3 tumors in vivo.
Data are presented as mean ± SD with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p <
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