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Tumor suppressor activity of RXR in AML
AB
CDE
Figure 6. All-trans retinoic acid (ATRA)/bexarotene in different leukemia cell lines and in human acute myeloid leukemia (AML) ex vivo. (A and B) Colony forming units (CFU) in methylcellulose per 2,000 Dnmt3a R878H/FLT3-ITD and Tet2-KO/FLT3-ITD leukemia cells treated as indicated for 7 days in duplicate. (C) Zip synergy score of ATRA and bexarotene interactions calculated by SynergyFinder.24 (D and E) Cell viability of primary human AML leukemia samples treated for 96 hours ex vivo with ATRA and bexarotene at indicated concentrations assessed in duplicate by CellTiter-Glo (CTG). Dx: sample acquired at initial diagnosis; R: sample acquired at relapse. *P<0.05, **P<0.01, t-test relative to control.
RARA ligands following transplantation of UAS-GFP x MLL-AF9 leukemia cells transduced with Gal4-RARA (Figure 1G), suggesting the absence of RARA ligands, in MLL-AF9 leukemia cells in vivo.
To determine whether natural RXRA ligands were pres- ent in other forms of murine leukemia, we repeated these studies in leukemias derived using activated Notch1 (T-cell leukemia)26 and TLS-ERG (erythroleukemia).27 In neither was the presence of mCherry+GFP+ cells observed (Figure 1H and I).
RXR acts as tumor suppressors in mouse MLL-AF9 leukemias
Given the presence of natural RXRA ligands in MLL- AF9 leukemia, we sought to determine whether activated endogenous mouse Rxrs contribute to leukemic growth in
vivo. We transduced Mx-Cre x Rxraflox/flox x Rxrbflox/flox BM cells with MLL-AF9 retrovirus, transplanted these into sublethally irradiated recipient mice, and derived subse- quent leukemias (see Figure 2A). Of note, Rxrg expression is low to absent in hematopoietic cells (Online Supplementary Figure S1G).28 We performed our initial retroviral transduction and transplantation in the absence of polyinosinic:polycytidylic acid (pIpC) treatment. However, low levels of native interferons induced Mx-Cre activity in a subset of cells, and a small proportion of BM cells exhibited Rxra and Rxrb deletion prior to transduction and transplantation (Figure 2B).23 Unexpectedly, all MLL- AF9-derived primary leukemias emerged with almost 100% deletion of both Rxra and Rxrb alleles even in the absence of any pIpC injections (Figure 2C), suggesting a strong positive selection for the D/D (deleted) alleles. In
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