br Figure A Induces Expression of
Figure 5. 673A Induces Expression of the Mitochondrial Uncoupling Proteins
(A) Count of viable PSB 1115 of CD133+ and CD133 PEO4 cells after 36-h treatment with transcription inhibitor flavopiridol ± 673A.
(C) qRT-PCR analysis of UCP1/3 expression 48 h after siRNA downregulation of ALDH1A family members in PEO4 cells.
(F) Quantification of basal and spare respiratory capacity (i) and ATP production in primary HGSC cells (ii) after treatment with 12.5 mM 673A as assessed by Seahorse.
Figure 6. The Effect of ALDH Inhibition on Tumor Stemness
(A) (i) Cell counts of SKOv3 cells over time after treatment with 1 mg/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.
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confirm inhibition of ALDH in tumors in vivo, we used isotopic tandem orthogonal proteolysis-activity based protein profiling (isoTOP-ABPP) and click chemistry (Speers and Cravatt, 2004; Weerapana et al., 2010) to evaluate ALDH1A enzyme activity. The 673A-treated tumors demonstrated clear protection of the catalytic residue Cys303 in ALDH1A1, whereas no binding to noncatalytic residues was detected (Figures 7Ii–ii and S5B). Finally, evaluation of tumors that progressed on 673A treatment demonstrated upregulation of ALDH activity, suggesting on-target resistance mechanisms (Figure S5Aiii).
In this study, we identified a selective, pan-ALDH1A family inhib-itor, 673A. This ALDH1Ai preferentially induced necroptosis in ovarian CSCs, and, with chemotherapy, 673A eliminates chemo-therapy-resistant tumors. This study links ALDH enzymatic activ-ity with the necroptosis pathway. Furthermore, it adds to the growing literature supporting ALDH enzymes as important ther-apeutic targets in cancer and demonstrates the efficacy of selec-tively targeting the ALDH1A family.
ALDH as a Therapeutic Target
ALDH1A isozymes are known to have a direct role in resistance to the chemotherapeutics ifosfamide and cyclophosphamide (Pinto et al., 2009). ALDH activity has also been broadly linked with resis-tance to chemotherapeutics such as paclitaxel and doxorubicin, which are not directly metabolized by ALDH isozymes (Bretz et al., 2012; Dylla et al., 2008). Numerous preclinical studies have tested the ability of ALDH inhibitors to reverse chemotherapy resistance. DSF has anti-cancer activity in vitro and in vivo (Irving and Daniel, 1987; Kast and Belda-Iniesta, 2009; Lin et al., 2011; Morrison et al., 2010; Rezk et al., 2015; Yip et al., 2011). Dimethyl ampal thiolester (DIMATE), an ALDH1 and ALDH3 inhibitor, depletes leukemia stem cells but, importantly, does not affect hematopoietic stem cells (Venton et al., 2016). More recently a potent ALDH1A1 selective inhibitor and a pan-ALDH1A inhibitor were found to synergize with chemotherapy (Huddle et al., 2018;
Yang et al., 2018), whereas an ALDH-targeted pro-drug effectively eliminated ALDHbright melanoma cells (Sarvi et al., 2018).
Clinical work also supports development of ALDHi. A phase IIb trial in patients with advanced lung cancer demonstrated a sta-tistically significant improvement in the overall survival of pa-tients treated with DSF and chemotherapy versus chemotherapy alone, with 10% of DSF-treated patients being disease free at 3 years (Nechushtan et al., 2015). Similarly, a small study of high-risk breast cancer patients receiving adjuvant chemotherapy and sodium diethyldithiocarbamic acid (a primary active metab-olite of DSF) revealed a trend toward increased overall and dis-ease-free survival (Dufour et al., 1993). These studies suggest not only the efficacy but also the safety of inhibiting ALDH activity in patients. Consistent with the safety of targeting ALDH1A fam-