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co-repressor (N-CoR) and the silencing mediator for retinoid and thyroid hormone receptors (SMRT) and recruiting histone deacetylases (HDAC). Local histone deacetylation then facilitates chromatin condensation and gene silencing. Ligand binding alters the heterodimer con- formation, displacing the co-repressors and facilitating binding of co-activator complexes composed by p160 family (TIF-2/SRC-1/RAC3) with histone acetylase activi- ty (HAT). Local histone acetylation then facilitates chro- matin decondensation and gene transcription activation.9,10
All-trans retinoic acid (ATRA) has been proposed as the natural ligand for RAR. Multiple natural ligands have been proposed for RXR, including modified retinoic acids (9-cis retinoic acid and 9-cis-13,14-dihydroretinoic acid) and long-chain fatty acids (C22:6, C22:5; C20:4, and C24:5), which are available in diverse tissues as well as in serum.11-15 Natural RXRA, but not RARA, ligands are present in nor- mal hematopoietic cells in vivo under steady-state condi- tions, with preferential distribution in myeloid cells (Gr1+) and following myeloid stress resulting from granulocyte- colony stimulating factor (GCSF) treatment.14
ATRA (tretinoin) has transformed the outcomes of M3-acute promyelocytic leukemia (APL).16,17 However, outcomes with ATRA in non-APL AML have yielded mixed results.18 Bexarotene is a pan-RXR-activating ligand, which is approved for the treatment of cutaneous T-cell lymphoma (CTCL).19 In small exploratory studies of non- APL AML, bexarotene demonstrated evidence of activity, but only in a small proportion of patients.20,21
We used an in vivo reporter assay to explore whether retinoid receptors in leukemia cells are transcriptionally active (and therefore might be best targeted with antago- nists) or transcriptionally inactive (and therefore might be best targeted with agonists). Our results show that natu- ral ligands for RXRA, but not RARA, are present at low levels in vivo in primary mouse myelomonocytic leukemia cells where they exhibit tumor suppressor phenotypes.
Methods
Hematopoietic cell culture
Mouse BM Kit+ cells were isolated using an Automacs Pro (Miltenyl Biotec, San Diego, CA, USA) and plated in RPMI 1640 medium, 15% fetal bovine serum (FBS), Scf (50 ng/mL), IL3 (10 ng/mL), Flt3 (25 ng/mL), Tpo (10 ng/mL), L-glutamine (2 mM), sodium pyruvate (1 mM), HEPES buffer (10 mM), penicillin/strep- tomycin (100 units/mL), β-mercaptoethanol (50 mM). MLL-AF9 leukemia was cultured in a similar media, but without Flt3, or Tpo. MOLM-13 were grown in RPMI1640 and 20% FBS; THP-1 in RPMI1640, 10% FBS, 0.05 mM MetOH; MONOMAC-6 in RPMI1640, 10% FBS, 2 mM L-glutamine, 2 mM NEAA, 1 mM sodium pyruvate, 10 ug/mL human insulin; OCI-AML-3 in α-MEM and 20% FBS. Fluorescence was detected on a FACS Scan, Gallios instrument (Beckman Coulter, Brea, CA, USA) or ZE5 Cell Analyzer (Biorad, Hercules, CA, USA).
Primary acute myeloid leukemia samples
All cryopreserved AML samples were collected as part of a study approved by the University of Helsinki after patients pro- vided informed consent in accordance with the Declaration of Helsinki. Thawed BM mononuclear cells were suspended in 87.5% RPMI 1640 medium plus 12.5% HS5 stromal cells condi- tioned media, and supplemented with 10% FBS, L-glutamine (2 mM) and penicillin/streptomycin (100 units/mL). 10,000 cells/well
Tumor suppressor activity of RXR in AML
in 25 mL were plated on 384-well plates containing bexarotene and ATRA. The cells were incubated with the drugs for 96 h after which cell viability was measured with CellTiter-Glo (Promega, Madison, WI, USA).
Mice
UAS-GFP and Mx1-Cre x Rxraflox/flox x Rxrbflox/flox mice were bred as previously described.22,23 pIpC treatment was intraperitoneal injection (IP) with 300 μg/mouse; four doses were given every other day. Rxra and Rxrb deletion were confirmed by polymerase chain reaction (PCR). Bexarotene was administrated by oral gav- age, suspended in sterile α-tocopherol suspension or in corn oil, 1 mg per mouse per day for 5 days/week. Lox-stop-Lox YFP mice were a gift from Todd Fehniger, Washington University. Dnmt3a- R878H/FLT3-ITD mouse leukemia cells were generated by Angela Verdoni and were a gift from Timothy Ley, Washington University (T Ley, unpublished material, 2019). To generate this line, hematopoietic cells derived from Dnmt3a R878H mosaic mice were transduced with an MSCV-FLT3-ITD-IRES-GFP retrovirus, and transplanted into multiple recipient mice. AML developing in these mice were confirmed to express the R878H allele by RNA- seq, with approximately 50% of all reads coming from the mutant allele (T Ley, personal communication 2019 ). The AML sample (AML- 1) examined in these studies is associated with rapid lethality in secondary transplants (median 48 days in syngeneic B6 animals) and an immature myeloid immunophenotype (Cd117+ and partial expression of Gr-1 and Cd11b) (O di Martino personal observation, 2020). Tet2-KO/FLT3-ITD leukemia cells were a gift from Ross Levine, Memorial Sloan Kettering Cancer Center. The Washington University Animal Studies Committee approved all animal exper- iments.
Study approval
All animal procedures were approved by the Institutional Animal Care and Use Committee of Washington University. All cryopreserved human AML samples were collected as part of a study approved by the University of Helsinki after patients pro- vided informed consent in accordance with the Declaration of Helsinki.
Results
RXRA natural ligands are present in MLL-AF9 myeloid leukemia cells in vivo
RARA and RXRA are preferentially expressed in mature myeloid cells and this pattern is reflected in a biased expression in M4/M5 AML where RARA and RXRA expression correlates with markers of maturation7 (Online Supplementary Figure S1). Therefore, we hypothesized that the availability of natural retinoids in hematopoietic malignancies might also be myeloid biased.
We evaluated in vivo retinoid ligand availability using a retroviral model of myelomonocytic leukemia and a reporter assay we had previously characterized.14,22 UAS-GFP bone marrow (BM) cells first were transduced with a retrovirus expressing MLL-AF9 and transplanted into sublethally irradiated recipient mice (see Figure 1A). Once leukemia emerged (i.e., UAS-GFP x MLL-AF9), these cells were transduced with a second retrovirus (MSCV-Flag-Gal4 DBD-RXRA LBD-IRES-mCherry) and subsequently transplanted into sublethally irradiated recipients. Using this strategy, leukemia cells with active, natural RXRA ligands express GFP (Online Supplementary Figure S2A and B). Ex vivo, the reporter was sensitive to
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