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Tumor suppressor activity of RXR in AML
culture (Figure 4). The relevant cell signals that influence retinoid responses in these two settings still have to be defined.
The synergy of ATRA and bexarotene can be under- stood within the context of co-repressor interactions with the RARA:RXRA heterodimer. Other studies of the RARA:RXRA heterodimer identified a subordinate role for RXR.32,39,41,42 Consistent with those results, mammalian two-hybrid assays demonstrated that RARA bound more effectively to co-repressors, that ATRA was more efficient than bexarotene at releasing co-repressors from RARA:RXRA, and that these limitations could be over- come using a bexarotene-derivative compound with dual RARA/RXRA activity (CW103-4) (Figure 5). Thus, the ATRA-dependent release of co-repressor from the RARA:RXRA heterodimer may account for the synergy observed between ATRA and bexarotene.
Within the RARA:RXRA heterodimer, specific RXRA domains appear necessary for anti-leukemic effects, including the AF1, DBD, and AF2, as well as amino acids responsible for ligand-binding and co-activator recruit- ment (Figure 3G). Thus, although ligand activation of RXR may be subordinate to RAR, RXR play an active, not a passive role in anti-leukemic response to ligands.
Other studies have suggested that biomarkers may identify subsets of retinoid-sensitive patients.54,55 We note that expression of RARA and RXRA are co-ordinately reg- ulated in AML with highest expression in M4/M5-FAB AML (Online Supplementary Figure S1). Cell lines and pri- mary AML samples suggest that combination retinoids may be active in leukemias beyond the mouse MLL-AF9 model and a potential bias in sensitivity within this group of patients (Figure 6). Larger studies will be required to accurately determine the specificity and sensitivity of these biomarkers as predictors of retinoid responses in non-APL AML. We have established an on-going ex vivo biomarker study of primary AML samples (clinicaltrials.gov identifier: NCT04263181) to further address this issue.
Although both ATRA and bexarotene are well-tolerat- ed orally-available compounds, approved by the US Food and Drug Administration, we found it difficult to provide mice with a sufficient dose to enable us to observe any big effects (Figure 7). Both oral ATRA and bexarotene are poorly soluble, have short serum half- lives (1 and 7 h, respectively), and their BM concentra- tions may be locally reduced by stroma expression of P450 enzymes.56 Given the short serum half-life of bexarotene, our administration of bexarotene (given every other day) is likely associated with a suboptimal area under the curve and incomplete receptor activation in vivo. Although ATRA is highly active against APL, administration and in vivo activity of ATRA in mouse models of APL have been surprisingly modest. For exam-
ple, across multiple mouse models, ATRA improved median survival in secondary transplants: 53 days versus 31 days,57 35 versus 25 days,58 approximately 90 days ver- sus approximately 50 days,59 approximately 75 days versus approximately 35 days,60 approximately 45 days versus approximately 35 days,61 and approximately 130 days versus approximately 85 days.49 In contrast, Westervelt et al. found that a liposomal formulation of ATRA (that is now no longer commercially available) resulted in much higher levels of ATRA in mice than ATRA slow-release pellets, and in 88% long-term sur- vival. Therefore, additional chemical or formulaic modi- fications may be required to optimize in vivo retinoids as anti-leukemic therapeutics, and the combination of free acid ATRA and bexarotene is not sufficient to overcome these limitations.
In summary, we find evidence of natural RXRA, but not RARA ligands in myelomonocytic MLL-AF9 mouse leukemia in vivo where the concurrent presence of recep- tors and active ligands acts as tumor suppressors. RARA and RXRA are co-ordinately expressed in myelomonocyt- ic leukemias, and optimal retinoid activation appears to require concurrent activation of both elements in the RARA:RXRA heterodimer, providing a further step toward improved retinoid therapeutics in AML.
Disclosures
No conflicts of interest to disclose.
Contributions
JSW and OdM designed experiments, performed experiments, and wrote the manuscript; HN, GH, HK, MAF, AV, JB, CW, MPM, MR and CH designed and performed experiments.
Acknowledgments
We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, MO, USA for the use of the Flow Cytometry Core. The Siteman Cancer Center is supported in part by an NCI Cancer Center Support Grant P30 CA91842. We thank High- Throughput Screening Center at Washington University School of Medicine in St. Louis, MO, USA. We thank Deborah Laflamme, Conner York, and Julie Richie for technical assistance.
Funding
This work was supported by NIH R01 HL128447 (to JSW), NIH P50 CA171963 (Project 1, to JSW and DRP) by the Siteman Investment Program (to JSW), and grants from the Spanish Ministerio de Ciencia e Innovación (MCI) (SAF2015- 71878-REDT-NurCaMeIn, RTI2018-095928-B100) (to MR). The CNIC is supported by the MCI and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV- 2015-0505).
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