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O. di Martino et al.
which contains a deletion of the RXRA C-terminal helix 12 (AF2 domain); this mutation is able to bind to RXR lig- ands, but does not transactivate the reporter (Figure 1C and E, and Online Supplementary Figure S2C).14
In vivo, a significative proportion of cells remained mCherry+GFP– (Figure 1B). To determine whether RXRA transactivation could be further stimulated, leukemic
RXRA reporter mice were orally gavaged for 2 days with 50 mg/kg bexarotene using a clinical formulation with improved solubility (Targretin). Bexarotene is a pan-RXR agonist and results were compared with vehicle control (water). We observed that bexarotene treatment further augmented RXRA-dependent GFP expression (Figure 1D and F). In contrast, we did not observe evidence of natural
ABC
DEF
G
Figure 3. Pharmacologic targeting of natural retinoic acid receptor (RAR)A and retinoid X receptor (RXR)A ligands blocks MLL-AF9 proliferation in vitro. (A) MLL- AF9 leukemia cells derived from UAS-GFP bone marrow (BM) and transduced with MSCV-Flag-Gal4-RXRA-IRES-mCherry retrovirus (MLL-AF9 Gal4-RXRA cells) were treated as indicated, replated after 48 hours (h), and total viable cells in 50 mL assessed in duplicate after 96 total h of treatment at indicated doses. (B) MLL-AF9 RXR-flox (wild-type, WT) or RXR-KO (Rxra/Rxrb deficient) leukemia cells (see Figure 2) were treated with all-trans retinoic acid (ATRA) and bexarotene for 96 total h and the synergy was calculated by SynergyFinder software24 using three different mathematical calculators for synergy versus additive effects (Zip, Bliss, and HAS). In these calculations, results >1 suggest mathematical synergy, although larger values are typically required for biologically relevant synergy. (C) MLL-AF9 Gal4-RXRA cells were treated as indicated, replated after 48 h, and total viable cells in 50 mL were assessed in duplicate after 96 total h of treatment. (D-F) MLL-AF9 leukemia cells were treated as indicated, replated after 48 h, and total viable cells in 50 mL assessed in duplicate after 96 h of total treatment. (G) Rxra/Rxrb deficient MLL- AF9 leukemia cells (RXR-KO) were transduced with retrovirus encoding MSCV-RXRA (full length)-IRES-mCherry or retrovirus with indicated RXRA mutations. Mutations and published mutation effects are indicated. 24 h after retroviral transduction, cells were treated in triplicate with 50 nM ATRA/bexarotene, and the pro- portion of mCherry+ cells was assessed relative to untreated control population after 72 h. **P<0.01, ***P<0.001, t-test with Welch’s correction relative to control.
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haematologica | 2021; 106(4)