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O. di Martino et al.
contrast, repeat experiments deriving Notch1 T-cell leukemia and TLS-ERG erythroleukemias showed near complete retention of Rxraflox/flox and Rxrbflox/flox alleles (Figure 2D and E). We performed secondary transplantation of MLL-AF9 leukemia cells derived from Mx-Cre x Rxraflox/flox x Rxrbflox/flox and from Rxraflox/flox x Rxrbflox/flox mice using 1x106 leukemia cells per recipient. Because these tumors were transplanted immediately, we were unaware of their dele- tion status and separately treated cohorts of mice with pIpC or control intending to determine whether the delet- ed allele augmented growth. We subsequently noted that primary tumors derived from Mx-Cre+ mice already car- ried deleted alleles and that these Rxra/Rxrb null (RXR-KO) leukemias resulted in shorter latency than tumors derived from Mx-Cre– mice, and that further stimulation by pIpC did not impact survival (Figure 2F). This suggests that RXR-KO leukemia cells grow more quickly in vivo than their wild-type counterparts, and could, therefore, have outgrown the wild-type populations during the primary transplantations. To exclude the possibility that MLL-AF9 leukemogenesis or hematopoietic transplantation could non-specifically activate Mx-Cre and induce deletion of the Rxr alleles, we crossed Mx-Cre mice with Lox-stop-Lox- YFP reporter mice and derived seven independent MLL-AF9 leukemias using three individual donors. The leukemia cells that emerged were not associated with increased YFP expression, suggesting that Mx-Cre activa- tion does not routinely occur following viral MLL-AF9 leukemogenesis and transplantation (Figure 2G).
Pharmacologic targeting RXRA and RARA in vitro
In vitro, bexarotene exerted dose-dependent growth inhibition in UAS-GFP x MLL-AF9 x Gal4-RXRA leukemia cells, although the effect was modest (2-4 fold) (Figure 3A, red line). Because Rxrs act as heterodimers with other orphan receptors, we screened for interactions with addi- tional ligands. The combination of bexarotene with either ATRA or tamibarotene (an RARA-specific agonist) lead to profound, dose-dependent growth inhibition while single- agent RARA ligands resulted in only modest (2-4 fold) growth inhibition (Figure 3A, blue and brown lines). Response and synergy were absent in RXR-KO MLL-AF9 leukemia cells (Figure 3B). The effect of ATRA on leukemic growth inhibition reached a plateau at 60-100 nM, whereas increasing concentrations of bexarotene were associated with increasing growth reduction up to 1 mM (Figure 3C). After 5 days of retinoids, we noted the proportion of GFP+ cells and the GFP median fluorescence intensity (MFI) was significantly reduced (Online Supplementary Figure S3A and B), suggesting that the most RXR responsive cells experienced the greatest negative
selection by retinoid treatment.
We tested a series of additional nuclear receptor ligands
for anti-leukemic activity. A broad range of RXR ligands cooperated with ATRA to induce anti-leukemic activity and again displayed only modest single-agent activity (Online Supplementary Figure S4A and B). The RARA-spe- cific agonist BMS753, but not the RARG-specific agonist BMS961, cooperated with bexarotene to inhibit MLL-AF9 cell growth (Figure 3D and E). RXR weak agonists and antagonists (LG100754 and HX531) were insufficient to cooperate with ATRA or tamibarotene (RARA specific agonist) and the pan-RAR inverse agonist BMS493 inhib- ited the effect of combination ATRA and bexarotene (Online Supplementary Figure S4C and D). Finally, the
PPARA agonist GW7647, PPARG agonist pioglitazone, and the LXR agonist GW3965 had modest effects with bexarotene (Figure 3F and Online Supplementary Figure S4E and F).
RXRA domains required for retinoid sensitivity
To map the structural domains of RXRA required for anti-leukemic activity, we retrovirally re-expressed RXRA in RXR-KO MLL-AF9 leukemia cells (Figure 3G). mCherry expression identified transduced cells. A series of RXRA deletions and mutations were generated and the position of the LBD mutations are highlighted within an available RXRA crystal structure (Protein Database: 4K4J) (Online Supplementary Figure S5A and B). The expression of the RXRA mutants was confirmed by western blot (Online Supplementary Figure S5C). In cells transduced with full- length RXRA, bexarotene and ATRA induced a strong decrease in the proportion of mCherry+ cells after 48 h (Figure 3G), consistent with a strong retinoid sensitivity among cells expressing wild-type RXRA. We found that mutants lacking the AF1, DBD, or AF2 domain and muta- tions reported to: disrupt the DNA binding zinc finger (E153G/G154S),29 reduce co-activator binding (L276A/V280A),30 or abrogate ligand binding (R316A/L326A),31 were unresponsive to retinoids (Figure 3G). Additional mutations reported to: reduce co-activator binding (E453A, V298A/L301A),32 reduce intracellular traf- ficking of RXRA (S260A),33 obstruct the heterodimer inter- face (Y402A, R426A)34 or reduce co-repressor binding to RXRA (K440E),32 largely retained sensitivity to retinoids (Figure 3G). These data demonstrate the necessity of spe- cific, functional RXRA domains (AF1, DBD, and AF2), as well as RXRA co-activator-interacting moieties for the activity of the RARA:RXRA heterodimer.
Maturation and apoptosis induced by ATRA/bexarotene
Retinoids have been associated with myeloid matura- tion programs.35-38 We observed that different culture con- ditions influenced the effect of retinoid-induced matura- tion and apoptosis in MLL-AF9 leukemia. When grown in methylcellulose, combination ATRA/bexarotene reduced colony-forming capacity in a dose-dependent manner (Figure 4A); these effects were associated with changes in the number of colonies, the size of the colonies (smaller), colony morphology (diffusion of cell clustering), and cellu- lar cytomorphology (acquisition of vacuoles), consistent with loss of self-renewal and maturation (Figure A-F). Again, these phenotypes were absent in RXR-KO leukemia cells and single-agent retinoids resulted in mod- est effects. However, when MLL-AF9 leukemia cells were grown in liquid culture, retinoid combinations did not induce cytomorphologic changes or cell cycle exit, but rather, they induced time- and dose-dependent apoptosis and activation of intrinsic caspase pathways (Caspases 3/7 and 9) (Figure 4G-L).
Co-repressor binding to RARA:RXRA heterodimers
The synergy observed between ATRA and bexarotene suggests different effects within the RARA:RXRA het- erodimer. In the absence of retinoids, the RARA:RXRA heterodimer binds to co-repressors such as the nuclear receptor co-repressor (NCOR1) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT, aka NCOR2).39-42 Using a mammalian two-hybrid assay in 293T cells,43 we noted that RARA resulted in greater tran-
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haematologica | 2021; 106(4)