Page 78 - 2022_02-Haematologica-web

O. di Martino et al.
Because the RXRA DT448/9PP-transduced cells acquired cell adhesion properties and ruffled borders suggestive of macrophage-like maturation, we assessed immunopheno- typic changes. Bexarotene treatment of RXRA WT trans- duced cells resulted in an increased percentage of cells expressing F4/80, CD11b, CD14, and CD64, as determined by flow cytometry, and increased the median fluorescence intensity (MFI) of F4/80, CD11b, CD14, CD64, Ly6c, and Gr1, but had little effect on CD115 expression. RXRA DT448/9PP transduction resulted in increased percentages of cells expressing CD11b, CD14, and CD64, with increased MFI of CD11b, CD14, CD64, Ly6c and Gr1, con- sistent with myeloid and monocytic maturation, but lack- ing the classical macrophage marker CD115 (Figure 1H, Online Supplementary Figure S1).
Transduction of RXRA DT448/9PP, but not RXRA WT into two KMT2A-MLLT3-associated human cells (THP1 and MonoMac-6) also resulted in adherence to plastic with the development of podocytes and ruffled borders (Online Supplementary Figure S2). These effects required 4 to 6 days to emerge, whereas adhesion and cell clumping in murine KMT2A-MLLT3 cells typically required only 3 or 4 days. In contrast, Kit+ murine bone marrow cells cultured in stem cell media (stem cell factor, interleukin 3, thrombopoietin, Flt3L) and K562 cells were resistant to the effects of RXRA DT448/9PP, and the cells remained round and non-adherent (Online Supplementary Figures S2D and S3A, B).
RXRA DT448/9PP is constitutively active
We assessed the functional activity of RXRA DT448/9PP using multiple reporter systems. First, we used a UAS/Gal4 reporter.11 KMT2A-MLLT3 WT leukemia cells derived from UAS-GFP bone marrow cells were retrovirally transduced with a fusion of the Gal4 DNA binding domain and the RXRA wild-type ligand-binding domain (Gal4-RXRA) or a fusion of the Gal4 DNA-binding domain and the RXRA DT448/9PP ligand-binding domain (Gal4-RXRA DT448/9PP). Gal4-RXRA DT448/9PP resulted in constitu- tive activity uninfluenced by increasing concentrations of bexarotene (Figure 2A). Constitutive activity of RXRA DT449/8PP was also observed in Kit+ UAS-GFP bone mar- row cells transduced with Gal4-RXRA retroviruses (Online Supplementary Figure S3C).
Second, we evaluated three different luciferase reporters in 293T cells. 293T cells are known to express endogenous RXRA as well as various of its partners; RARA, PPARG, and VDR.20 Reporters assays included a synthetic direct repeat 1 (DR1) peroxisome proliferator response element (PPRE); a DR1 response element from the ApoA1 promoter; and a DR5 response element from the RARB promoter. We noted that RXRA DT448/9PP transfection consistently led to lig- and-independent activation of all three constructs (Figure 2B). A control RXRA construct contained a deletion of the AF2 domain (RXRA DAF2), and this construct is unrespon- sive to ligand. Using this approach we assessed whether RXRA DT448/9PP might be sensitive to inhibition by two pan-RXR antagonists, HX531 and UVI3003. Neither com- pound inhibited activation of the UAS/Gal4 reporter or the RARE-Luc reporter (Figure 2C).
We used a mammalian two-hybrid assay to assess the interaction of RXRA DT448/9PP with the co-activator PGC1a, noting constitutive, ligand-independent binding (Figure 2D, E). This activity remained present with the L2/3A variant (L147A, L148A, L210A, L211A),21 which con- tains point mutations in the critical LXXLL motifs required
for ligand-dependent interactions of PGC1a with nuclear receptors. Thus, RXRA DT448/9PP appears to constitutive- ly engage PGC1a using domains outside the canonical N- terminal LXXLL motifs.
Comparison with other active RXRA variants
Two other RXRA variants with constitutively active prop- erties have been described. First, mouse Rxra F318A exhibits constitutively active phenotypes.22 However, when a crystal structure was generated, the ligand-binding pocket con- tained oleic acid, and Rxra F318A activity could be inhibited by the pan-RXR antagonist HX531, suggesting that the mutation leads to augmented responsiveness to a natural lig- and present in the tissue culture, and is not completely lig- and-independent.23
We assessed the sensitivity of RXRA DT448/9PP to two pan-RXR antagonists (HX531 and UVI3003) across a series of assays. In RXR-WT KMT2A-MLLT3 leukemia cells trans- duced with RXRA DT448/9PP, cell adhesion, clumping, and loss of proliferation phenotypes were not abrogated by either compound (Online Supplementary Figure S4).
To further assess whether RXRA DT448/9PP pheno- types may result from hyper-responsiveness to intracellu- lar natural ligands, we mutated two amino acids (R316 and L326) that form critical ionic bonds with the car- boxylic acid group in ligands (e.g., bexarotene, 9-cis retinoic acid, and long-chain fatty acids).5 RXRA R316A/L326A has been previously shown to abrogate lig- and-dependent activation and is unable to rescue response to bexarotene in RXR-KO KMT2A-MLLT3 leukemia cells.5,24 Retroviral expression of the compound variant RXRA R316A/L326A/DT448/9PP again led to overexpres- sion on western blot and resistance to proteolytic cleav- age, but did not abrogate cell clumping, loss of prolifera- tion, or loss of colony formation (Figure 3A-D).
Second, recurrent RXRA hot-spot mutations (S427F/Y) occur in patients with bladder cancer, and these augment the activity of the PPARG:RXRA heterodimer, but are not capa- ble of independently activating RXRA reporters.25 Retroviral expression of RXRA S427F also led to strong overexpression of the variant, which retained sensitivity to proteolytic cleavage, although cell adhesion, cell clumping, cell prolifer- ation, and colony-forming properties were not altered (Figure 3E-H).
To determine whether similar heterodimerization with PPARG or with other nuclear receptors may play essential roles in the activity of RXRA DT448/9PP, we evaluated phe- notypes using a series of receptor antagonists. Concurrent treatment with potent antagonists of retinoic acid receptors, LXR, PPARA, and PPARG did not affect clumping, adhesion to plastic, or proliferation phenotypes (Online Supplementary Figure S5), demonstrating that the DT448/9PP creates a non- permissive receptor that is functionally active, independent- ly of other activated nuclear receptors.
RXRA DT448/9PP inhibits leukemic engraftment and expansion
KMT2A-MLLT3 WT cells were transduced with RXRA WT or RXRA DT448/9PP retroviruses labeled with IRES- mCherry cassettes (Figure 4A). To limit maturation effects in vitro, the populations were immediately transplanted into recipient mice and residual cells were subsequently ana- lyzed by flow cytometry. After 4 weeks of engraftment and expansion, leukemia cells in the peripheral blood were assessed and we observed that mice transplanted with
420
haematologica | 2022; 107(2)