br ily members ALDH A is dispensable for the
ily members, ALDH1A1 is dispensable for the function of murine stem cells (Levi et al., 2009).
Unfortunately, DSF is not an ideal ALDH inhibitor for cancer therapy: DSF has a very short half-life in vivo and numerous active metabolites that are associated with off-target effects, increased toxicity, and dose-limiting specificity (Agarwal et al., 1986; Mal-colm et al., 2008; Petersen, 1992). In addition, although DSF can partially reverse chemotherapy resistance in ovarian cancer cells, it has limited efficacy in ovarian cancer in vivo (Rezk et al., 2015). Our analysis of ovarian cancer suggests that different can-cers may express different ALDH1A family members. Indeed, ALDH1A1, ALDH1A2, and ALDH1A3 are all reported to be ex-pressed in ovarian cancer (Saw et al., 2012). We observed that siRNA KD or CRISPR KO of one ALDH1A was associated with reduced, but not complete, loss of response to ALDH1Ai. We believe this is likely related to expression of compensatory ALDH1A family members. Dual KD/KO experiments will be necessary to address this, but these are technically challenging because of difficulties in maximizing KD of multiple genes without increasing toxicity related to nucleic SCH 58261 concentrations. Multi-KO CRISPR is an alternative but is more time consuming, requires multiple selection methods or steps, and has the risk of off-target genome editing (Fu et al., 2013).
Given strong support in the literature for a therapeutic role of ALDH1A enzymes in chemotherapy resistant and stemness, var-iable expression of different ALDH1A family members in can-cers, and the potential redundancy of function for these ALDH1A family members, we developed a pan-ALDH1A family member inhibitor. We found that the ALDH1A pan-family inhibitor 673A effectively reversed chemotherapy resistance. The 673A was effective in cell lines that predominantly expressed either ALDH1A1 or ALDH1A3. Importantly, in several in vivo models of ovarian cancer, 673A significantly enhanced chemothera-peutic activity, resulting in tumor eradication.
The Effect of Targeting CSCs
Although there remains controversy regarding CSCs, ALDH enzymatic activity, either alone or in combination with other stem cell markers, is arguably the best-characterized CSC marker. In ovarian cancer, ALDH+/CD133+ ovarian cancer CSCs meet the definition of CSCs put forth by Pattabiraman and Weinberg (2014); ALDH+/CD133+ ovarian cancer cells demonstrate both (1) increased ability to initiate tumors (Silva et al., 2011) and (2) a clear ability to undergo asymmetric divi-sion to both self-renew and produce non-self-daughter cells (Choi et al., 2015). The 673A preferentially induced cell death in CD133+ ovarian cancer cells. Despite targeting less than 10% of the cells in most cancer cell lines, 673A had a signifi-cant effect on tumor growth in vivo. As a single agent, 673A could reduce tumor growth >50%. As noted above, when com-bined with chemotherapy, 673A increased tumor eradication rates in multiple in vivo models of ovarian cancer. We believe this is both via the direct targeting of CD133+ CSCs and the
(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.
(E) Percentage of mice that were tumor free 6 months after inoculation with 1 3 105 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.
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reversal of ALDH1A-driven chemotherapy resistance. Taken together, these data support a critical role for CSCs, whether inherent or because of de-differentiation, as a therapeutic target.