Page 83 - 2022_02-Haematologica-web

RXRA active mutation
analysis, but manual curation found that the most of these 11 transcripts are related to immune function and activity.
Discussion
Retinoic X receptors and retinoic acid receptors have been shown to influence myeloid maturation.1,2,4,27,28 RXRA protein levels and activity have been linked to cell fate deci- sions at the neutrophil versus monocyte decision,29 and dele- tion of Rxra and Rxrb prevents osteoclast maturation.12 and augments KMT2A-MLLT3 leukemogenesis and cell expan- sion in vivo,5 suggesting a role for retinoic X receptors in reg- ulating myeloid maturation.
RXRA DT448/9PP is a serendipitously discovered muta- tion that results in constitutive activity, leukemic cell matu- ration, and loss of proliferative capacity. This variant demonstrates ligand-independent activation, augmented co-activator binding, induced maturation transcripts by RNA sequencing, and reduced engraftment in vivo. To our knowledge, RXRA DT448/9PP has not been spontaneously observed in cancer or other pathological states.
Two other RXRA mutations with activating phenotypes have been described. Mouse Rxra F318A has increased tran- scriptional activity, potentially via increased responsiveness to cell-available oleic acid.22 However, Rxra F318A retains sensitivity to RXRA antagonists, whereas RXRA DT448/9PP did not (Figure 2C, Online Supplementary Figure S2). Furthermore, R316 and L326 are critical amino acids that interact directly with bound ligand and are required for ligand activation of RXRA.5,24 Combining these ligand- blocking mutations with DT448/9PP, we noted retained KMT2A-MLLT3 maturation phenotypes (Figure 3A-C), fur- ther suggesting ligand-independent activity of RXRA DT448/9PP. Recurrent RXRA hot-spot mutations (S427F/Y) have been noted in patients with bladder cancer. These mutations augment the activity of the PPARG:RXRA het- erodimer and are not capable of independently activating RXRA in reporter assays.25 We previously found that com- binations of RARA and RXRA ligands lead to leukemic maturation and apoptosis, whereas PPARG and RXRA lig- ands did not.5 Here we found that RXRA S427F did not recapitulate leukemia maturation phenotypes in KMT2A- MLLT3 leukemia cells (Figure 3E-G), and the PPARG antag- onist T0070907 did not abrogate RXRA DT448/9PP pheno- types (Online Supplementary Figure S5). Thus, in contrast to Rxra F318A and RXRXA S427F, RXRA DT448/9PP may be constitutively active, independently of endogenous, avail- able, natural ligands, and also may not depend on activation through PPARG:RXR heterodimers or other major nuclear receptor heterodimers.
RXRA DT448/9PP constitutive activity may be related to at least two phenotypes. RXRA DT448/9PP is resistant to enzymatic cleavage, and this may enable augmented func- tional protein levels (Figures 1F and 3D, H), but cannot explain resistance to pan-RXR antagonists or co-mutation with R316A/L326A (Figures 2C and 3A-C, Online Supplementary Figure S4). RXRA DT448/9PP was also asso- ciated with ligand-independent co-activator binding (Figure 2D), which may enable augmented, or even constitutive activity. A limitation of these studies is that they require a retroviral overexpression system and therefore the effect of DT448/9PP RXRA at physiological levels is unknown.
Not all cells tested were susceptible to RXRA DT448/9PP. The two human myelomonocytic acute myeloid leukemia
lines with KMT2A-MLLT3 (THP1 and MonoMac-6) were susceptible, whereas the heterogeneous stem/progenitor populations of Kit+ murine bone marrow cells and the blast phase chronic myeloid leukemia cell line K562 were not (Online Supplementary Figures S2 and S3). Maintained in stem cell cytokines, Kit+ bone marrow cells have multipo- tent potential and their medium lacks cytokines that might provide monocytic maturation support. K562 cells are sus- ceptible to erythroid maturation stimuli rather than myeloid maturation. Other groups have observed differ- ences in sensitivity and resistance to retinoids across cell lines.9,30,31 Thus, different external signals and/or internal priming states may affect the susceptibility to retinoids and to RXRA DT448/9PP.
We have previously noted that Rxra and Rxrb expression negatively regulates KMT2A-MLLT3 leukemia and that these cells are exposed to low levels of natural RXRA lig- ands in vivo.5,32 Similarly, here we found that cells that over- express RXRA WT consistently engraft in recipient mice, but are associated with a competitive disadvantage relative to untransduced, mCherry-negative cells (Figure 4). This phenotype was augmented by transduction with RXRA DT448/9PP, which further limited engraftment and leukemic outgrowth in vivo, again suggesting the potential of activated retinoic X receptors to inhibit growth of leukemia cells in vivo.
RXRA DT448/9PP resulted in several maturation-related phenotypes, including lack of proliferation, loss of colony formation, acquisition of ruffled borders and podocytes, and increased expression of cell surface markers of myeloid and monocytic maturation. Transcriptional analysis of bexarotene-treated cells versus RXRA DT448/9PP-trans- duced cells suggested strong overlapping myeloid and monocytic maturation signatures, consistent with ligand- independent, constitutively active effects of DT448/9PP. Many maturation-related transcripts were more effectively induced by RXRA DT448/9PP than by bexarotene, and a few myeloid-related transcripts were uniquely induced by RXRA DT448/9PP. Thus, DT448/9PP may more effectively activate the same loci as ligand-activated RXRA, and activ- ity at select novel loci may enable additional phenotypes.
Like other nuclear receptors, the retinoic X receptors are ligand-dependent transcription factors and their function and activity change from transcriptional repressors to tran- scriptional activators in the presence of active ligands. Multiple natural ligands have been proposed for the retinoic X receptors,6 and it is often difficult to know which cells and settings contain active, natural ligands, and when an observed effect of retinoic X receptors may be related to the receptor function in the absence versus presence of ligand. A constitutively active variant provides a helpful genetic com- parator and could be used with diverse forms of previously characterized non-functional (e.g., DDBD, E153G/G154S) and ligand-non-responsive variants (e.g., DAF2, R316A/326A).
Retinoid receptors have been an attractive therapeutic target in acute myeloid leukemia; however their effects in clinical trials have been modest. The maturation effects (loss of proliferation and colony-forming potential, and morphological changes) of DT448/9PP on KMT2A-MLLT3 leukemia cells are more robust than the maximal effects of retinoid ligands such as all-trans retinoic acid and bexarotene. Although further delineation of mechanistic activity and cell-type susceptibility rules will be required, these data suggest that current small-molecule retinoids
haematologica | 2022; 107(2)
425