Uncoupling RARA transcriptional activation and degradation clarifies the bases for APL response to therapies

Abstract

In PML/RARA-driven acute promyelocytic leukemia (APL), retinoic acid (RA) induces leukemia cell differentiation and transiently clears the disease. Molecularly, RA activates PML/RARA-dependent transcription and also initiates its proteasome-mediated degradation. In contrast, arsenic, the other potent anti-APL therapy, only induces PML/RARA degradation by specifically targeting its PML moiety. The respective contributions of RA-triggered transcriptional activation and proteolysis to clinical response remain disputed. Here, we identify synthetic retinoids that potently activate RARA- or PML/RARA-dependent transcription, but fail to down-regulate RARA or PML/RARA protein levels. Similar to RA, these uncoupled retinoids elicit terminal differentiation, but unexpectedly fail to impair leukemia-initiating activity of PML/RARA-transformed cells ex vivo or in vivo. Accordingly, the survival benefit conferred by uncoupled retinoids in APL mice is dramatically lower than the one provided by RA. Differentiated APL blasts sorted from uncoupled retinoid-treated mice retain PML/RARA expression and reinitiate APL in secondary transplants. Thus, differentiation is insufficient for APL eradication, whereas PML/RARA loss is essential. These observations unify the modes of action of RA and arsenic and shed light on the potency of their combination in mice or patients.

Figure 1.

Ligand-induced RARA transactivation and degradation can be uncoupled and control distinct biological effects. (A) Structures of RA, etretinate (Etre), acitretin (Aci) and NRX195183 (NRX). (B) Western blot analysis of RARA-transduced rars^−/− MEFs after 6 h of treatment with RA, acitretin, or NRX at 1 µM. Actin demonstrates equal loading. (C) RA, acitretin, or NRX (1 µM), activate cyp26 and rarb gene expression in rars^−/− MEFs stably reexpressing RARA, mean ± SD of three independent experiments. (D) Dose–response analysis of RA, acitretin, or NRX for the activation of RARE3-TK-Luc, a luciferase RA responsive reporter gene, in RARA-transduced rars^−/− MEFs. Results are expressed as percentage of maximal activation, demonstrating similar affinities of the three retinoids. Means ± SD of 3 independent experiments. (E) Correlation between RA-, acitretin-, and NRX-triggered gene activation in rars^−/− MEFs stably reexpressing RARA and treated for 8 h. Logarithmic scale. Analysis was restricted to genes induced more than twofold by RA or uncoupled retinoids in the transcriptomic arrays. Linear regression of the data is shown. Mean values of two independent experimental replicates. (F) Effect of RA, acitretin, or NRX on colony formation of RARA-transformed lineage-negative (Lin⁻) primary mouse hematopoietic progenitors after the first plating in methylcellulose (means ± SD of 3 independent experiments). (G) Effect of RA, acitretin, or NRX treatment on the cells described in F after 1 wk of methylcellulose culture (May–Grunwald Giemsa [MGG] staining, top; bar, 10 µm) or 24 h in liquid culture (FACS analysis, log₁₀ fluorescence, bottom). Representative experiment of three independent ones.

Figure 2.

Ligand-induced PML/RARA transactivation and degradation result in distinct biological effects. (A) RA, acitretin, or NRX (1 µM) activate rarb and tgm2 gene expressions in rars^−/− MEFs stably expressing PML/RARA. Mean ± SD of three independent experiments. (B–D) Western blot analysis (B), differentiation on MGG staining (C, top; bar, 10 µm), and colony formation (D) of PML-RARA-transformed Lin⁻ murine hematopoietic progenitors after 7-d treatment in methylcellulose culture with RA, acitretin, or NRX at 1 µM. Differentiation by FACS analysis (log₁₀ fluorescence) after 24 h of treatment in liquid culture (C, bottom). Representative experiment of three independent ones.

Figure 3.

Uncoupled retinoids fail to clear APL. (A) Survival analysis of APL mice treated for 7 d with RA, etretinate (acitretin precursor), or NRX. 5 mice per group for NRX or etretinate, 10 for RA and controls. Pooled data from two independent experiments. Log rank test was used to evaluate significance of the survival difference between APL mice treated with RA or uncoupled retinoids. (B) Circulating levels of RA, acitretin, or NRX 3 h after initiation of therapy. Mean values of two mice per condition. (C) Correlation between RA-, etretinate-, and NRX-triggered gene activation in APL cells treated for 6 h in vivo. Logarithmic scale. Analysis was restricted to genes induced more than twofold by RA in the transcriptomic arrays. Linear regression of the data are shown. Mean value of two mice per condition. (D) MGG staining (bar, 10 µm) and FACS analysis (log₁₀ fluorescence) of bone marrow cells from APL mice treated with RA, acitretin, or NRX for 3 d. Representative experiment of three independent ones. (E) Western blot analysis of PML/RARA levels in APL cells after 3-d in vivo treatment. (F) Quantification by Q-PCR of PML/RARA DNA in bone marrow cells of APL mice after 3- or 6-d treatment with RA, etretinate, or NRX. Means ± SD of three mice per condition. (G) Survival of secondary recipients inoculated with 10⁶ bone marrow cells from 3- or 6-d treated APL mice. 10 mice per group for NRX or etretinate, 20 for RA and controls at 3 d. 8 per group at 6 d. Pooled data from 3 independent experiments. Log rank test was used to evaluate significance of the survival difference between secondary recipients injected with bone marrow cells from RA- or uncoupled retinoid–treated APL mice.

Figure 4.

APL transplantation by inoculation of PML/RARA-expressing, terminally differentiated APL cells. (A) FACS sorting of the Mac-1^high/Gr-1^high cells from the bone marrow of APL mice treated for 3 d with RA, NRX, or acitretin (log₁₀ fluorescence). (B) Immunofluorescence analysis of PML reveals nuclear PML/RARA microspeckles in bone marrow cells from 3-d NRX- or etretinate-treated mice (bar, 10 µm). Representative cell obtained in two different experiments. (C) Death by APL of sublethally irradiated syngeneic recipients inoculated with cells (10³ or 10⁴) sorted in A.