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Tumor suppressor activity of RXR in AML
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Figure 7. Concurrent all-trans retinoic acid (ATRA)/bexarotene treatment reduces MLL-AF9 leukemia burden in vivo. (A) Schema for leukemia transplant procedure and in vivo treatment. Sublethally irradiated FVB mice were transplanted with 0.5x106 MLL-AF9 leukemia cells. Ten days later the mice were divided in four different cohorts and treated with 21-day release ATRA pellets. 5 mg: n=5 (~0.23 mg/day), 10 mg: n=5 (~0.5 mg/day), and 25 mg: n=8 (~1.2 mg/day). One day after pellet implantation, bexarotene (50 mg/kg) was delivered by oral gavage as indicated. A control cohort was implanted with placebo pellets and treated with the vehicle gav- age (n=12). IV: intravenous. (B) The mice were sacrificed 21 days from pellet implantation and the spleen weight analyzed. (C) Kaplan-Meier survival curve analysis of mice transplanted with MLL-AF9 cells and treated as indicated in (A). (D) Schema for leukemia transplant procedure and mice treatment. FVB mice were trans- planted with 1.5x106 MLL-AF9 leukemia cells by intraperitoneal (IP) injection. Five days later the mice were divided in four different cohorts and implanted with 21- day release ATRA pellets 25 mg (~1.2 mg/day) or placebo pellets. One day after pellet implantation, bexarotene (Targretin, 50 mg/kg) was delivered by oral gavage as indicated. (E) Kaplan-Meier survival curve analysis of mice transplanted with MLL-AF9 cells and treated as indicated in (E). *P<0.05, **P<0.01, ***P<0.001, t-test.
scriptional activity with either co-repressor (SMRT) versus RXRA, suggesting a stronger affinity of RARA to co- repressors than RXRA (Figure 5A). Similarly, when the bait and prey strategy was reversed (Figure 5B), ATRA led to greater reporter inhibition than bexarotene, with as great an effect as the combination of ATRA and bexarotene (Figure 5B). Similar results were noted using NCOR as bait (Figure 5C), suggesting that co-repressor interactions with RARA:RXRA heterodimers are dominat- ed by interactions with RARA.
A bexarotene derivative (CW103-4) had been identified with potential dual RARA/RXRA activity.44-46 CW103-4 has improved murine pharmacokinetics compared with bexarotene (peak plasma concentration of 152,955.83 vs. 18,633.33 ng/mL and area under the curve of 51,531 vs. 8,523 ng/mL).43 However, this is associated with a 5-fold increase in triglycerides 24 h after treatment,43 making it an interesting tool compound, but not an obvious clinical therapy. We assessed this compound to determine
whether its dual-affinity might provide single-agent activ- ity in vitro. Indeed, CW103-4 exhibited dual RARA and RXRA activation in UAS-GFP x MLL-AF9 leukemia cells, and was capable of ATRA-independent anti-leukemic activity (Figure 5D-F and Online Supplementary Figure S6A- D). Specifically, CW103-4 induced proliferative capacity reduction and apoptosis mediated by caspases 3/7 activa- tion (Online Supplementary Figure S6A-D). Interestingly, CW103-4 also induced consistent single-agent co-repres- sor release (Figure 5G). To determine whether these effects might occur through LXRs or PPARD, we assessed reporter activation and found no evidence of cross-reactiv- ity with these receptors (Online Supplementary Figure S6E- G). These results suggest that single-agent bexarotene is ineffective to induce co-repressor release from the RARA:RXRA heterodimer, and that co-repressor release results primarily from RARA activation, providing an explanation for the anti-leukemic combination synergy (Figure 3A and B).
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