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O. di Martino et al.
in vivo engraftment, and RNA sequencing, and present evi- dence of ligand-independent loss of proliferative capacity, transcriptional signatures of myeloid maturation that over- lap with signatures induced by the potent RXR ligand bexarotene, and inhibition of engraftment and leukemic expansion in vivo. These data again suggest that programs of leukemic cell growth and maturation may be susceptible to retinoids and provide a novel constitutively active tool for further delineating the function and activity of retinoid receptors.
Methods
Mice
Mice were maintained in a specific pathogen-free barrier facility with a 12 h light-dark cycle. Upon weaning, all mice were housed in groups of up to five mice of the same sex per cage. Food and a water bottle were provided in a recess of the metal wire lid at the top of the cage. Cages were changed once every week. Six- to ten- week-old mice (C57Bl/6 background) were typically used for the experiments. Equal numbers of male and female mice were used; no gender biases were noted.
Hematopoietic cell culture
Murine bone marrow Kit+ cells were isolated using an Automacs Pro (Miltenyl Biotec, San Diego, CA, USA) according to the manufacturer’s protocol. Kit+ cells were plated in progenitor expansion medium (RPMI 1640 medium, 15% fetal bovine serum, stem factor [50 ng/mL], interleukin 3 [10 ng/mL], Flt3L [25 ng/mL], thrombopoietin [10 ng/mL], L-glutamine [2 mM], sodium pyru- vate [1 mM], HEPES buffer [10 mM], penicillin/streptomycin [100 units/mL], b-mercaptoethanol [50 mM]) overnight and transduced with MSCV-KMT2A-MLLT3 retrovirus by spinfection with 10 μg/mL polybrene and 10 mM HEPES at 2400 rpm, 30°C for 90 min in an Eppendorf 5810R centrifuge. Cells were transplanted into sublethally irradiated mice and subsequent leukemia harvested 4 to 6 months later. KMT2A-MLLT3 leukemia cells were cultured in vitro using similar media, but without Flt3L or thrombopoietin. KMT2A-MLLT3-RXR-knockout (KO) leukemia cells were derived from Mx1-Cre x Rxraflox/flox x Rxrbflox/flox bone marrow cells11,12 trans- duced with MSCV-KMT2A-MLLT3 retrovirus and generated as described above. RXR deletion was induced by injecting Mx1-Cre x Rxraflox/flox x Rxrbflox/flox mice intraperitoneally with pIpC 300 mg/mouse; four doses were given, every other day. RXR deletion was confirmed by polymerase chain reaction analysis 4 weeks after mice had been treated with pIpC. THP1, and K562 cells were obtained from the American Type Culture Collection. Monomac- 6 and OCI-AML3 cells were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen.
RNA sequencing
KMT2A-MLLT3 leukemia cells were transduced with MSCV- RXRA-IRES-mCherry or MSCV-RXRA DT448/9PP-IRES-mCherry and treated with or without 250 nM bexarotene. After 24 h, mCherry+ cells were sorted and total RNA was extracted with TRIzol LS (Ambion Life Technologies) and isolated on PureLink RNA Kit columns (Thermo Fisher Scientific). The quality of RNA was measured on a 2100 Bioanalyzer (Agilent). Sequencing libraries, each with individual Illumina indexes, were constructed using the TruSeq Stranded mRNA procedure (Sample Prep Kit v2; Illumina). Libraries were sequenced as paired-end 151 bp reads on an Illumina NovaSeq instrument. Reads were aligned to the mm10 mouse reference genome, and transcription was quantified using kallisto version 0.43.1 13 and Ensembl transcripts version 95. The R
package edgeR was used to determine genes that were differen- tially expressed between groups: RXRA WT cells, RXRA WT cells treated with bexarotene , and RXRA DT448/9PP-transduced cells. Differentially expressed genes were defined as having an absolute log2 fold change >2 and a P-value <0.0001. Functional pathway analysis was performed using Panther Gene Ontology Analysis software14 with a Fisher exact test using the Bonferroni correction for multiple testing. Pathways were called if P<0.05 and fold enrichment was >2. Gene set enrichment analysis was performed using gene sets curated in the MSigDB database and GSEA 4.0.3 software; RRID:SCR_003199.15
Study approval
All animal procedures were approved by the Institutional Animal Care and Use Committee of Washington University.
Results
Effects of RXRA DT448/9PP on maturation
We recently evaluated a series of RXRA truncations and mutations to map the domains of RXRA required for anti- leukemic effects in murine KMT2A-MLLT3 (formerly MLL- AF9) leukemia cells derived from Mx-Cre x Rxraflox/flox x Rxrbflox/flox bone marrow cells (RXR-KO KMT2A-MLLT3 leukemia).5 Ghosh et al. had previously observed that RXRA DT448/9PP was associated with increased binding to an NCOR2 co-repressor (nuclear receptor corepressor 2, for- merly SMRT) peptide in a GST pull-down assay.16 We included this variant as part of the screen to evaluate co- repressor requirements. DT448/9 is positioned at the end of the turn between helix 11 and 12. Helix 11 directly binds to co-repressors and co-activators.17 Helix 12 (the AF2 domain) adopts an extended and unstructured conformation in the absence of ligand and assumes a repositioned and helical conformation following ligand binding (Figure 1A).18 Unexpectedly, retroviral expression of RXRA DT448/9PP (MSCV-RXRA DT448/9PP-IRES-mCherry) in RXR-KO KMT2A-MLLT3 leukemia cells induced the normally round cells to form adherent clumps that were mCherry+ on the tissue culture plate (Figure 1B). mCherry+ cells were also associated with loss of leukemic colony-forming capacity, lack of proliferation, and macrophage-like cytomorphology with ruffled borders and vacuoles (Figure 1C-E). These effects were absent in RXR-KO KMT2A-MLLT3 cells retro- virally transduced with RXRA WT (MSCV-RXRA-IRES- mCherry), with other tested RXRA domain deletions or point mutations, or with treatment combinations of RXRA and RARA activating ligands5 (Figure 1B). In addition, we found that retroviral expression of RXRA DT448/9PP (MSCV-RXRA DT448/9PP-IRES-mCherry) in KMT2A- MLLT3 WT leukemia cells induced the same phenotype as that observed in RXR-KO KMT2A-MLLT3 leukemia cells (lack of proliferation and macrophage-like cytomorpholo- gy) (data not shown). We examined the resulting proteins on western blot after transduction into KMT2A-MLLT3 WT cells (Figures 1F). RXRA DT448/99PP was associated with a similar sized band as wild-type RXRA, but it appeared resistant to the proteolytic cleavage that results in the for- mation of a truncated 36 kDa fragment.19 Endogenous Rxra levels were significantly lower than levels of retrovirally expressed RXRA (Figure 1G). Thus, retroviral transduction led to similar levels of expression of RXRA WT and RXRA DT448/9PP, at supraphysiological protein levels compared to endogenous RXRA.
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