In vivo analysis of the role of aberrant histone deacetylase recruitment and RAR alpha blockade in the pathogenesis of acute promyelocytic leukemia

Abstract

The promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) protein of acute promyelocytic leukemia (APL) is oncogenic in vivo. It has been hypothesized that the ability of PML-RARalpha to inhibit RARalpha function through PML-dependent aberrant recruitment of histone deacetylases (HDACs) and chromatin remodeling is the key initiating event for leukemogenesis. To elucidate the role of HDAC in this process, we have generated HDAC1-RARalpha fusion proteins and tested their activity and oncogenicity in vitro and in vivo in transgenic mice (TM). In parallel, we studied the in vivo leukemogenic potential of dominant negative (DN) and truncated RARalpha mutants, as well as that of PML-RARalpha mutants that are insensitive to retinoic acid. Surprisingly, although HDAC1-RARalpha did act as a bona fide DN RARalpha mutant in cellular in vitro and in cell culture, this fusion protein, as well as other DN RARalpha mutants, did not cause a block in myeloid differentiation in vivo in TM and were not leukemogenic. Comparative analysis of these TM and of TM/PML(-/-) and p53(-/-) compound mutants lends support to a model by which the RARalpha and PML blockade is necessary, but not sufficient, for leukemogenesis and the PML domain of the fusion protein provides unique functions that are required for leukemia initiation.

Figure 1.

Generation of the mutant RARα transgenic mice. (A) Mutant RARα cDNAs were cloned into the SalI site of the hCG expression cassette. Shaded boxes: PML and HDAC1 sequences. Capital letters: RARα domains. A schematic representation of the hCG is provided at the bottom of panel A. The regions flanking the 5′ and 3′ of the polylinker are indicated (5′ FL and 3′ FL, respectively). The 5′ FL region comprises the hCG promoter. White boxes: exons. Restriction endonuclease sites are indicated. CT: probe for Southern blotting. (B) Southern blot of genomic DNA from transgenic founders digested with EcoRI and hybridized with probe CT. The transgene examined is indicated on the left side of the panel. Probes for the single copy genes p62^DOK or PLZF were used as internal standards. WT, wild type. The numbers above the individual panels indicate the founder lines. (C) RT-PCR analysis of RARα mutant mRNA extracted from bone marrow cells. RT, reverse transcriptase.

Figure 2.

Biochemical properties of HDAC1-RARα. (A) HDAC1-RARα homo- and heterodimerizes in vivo. 293T cells were transfected as indicated. Immunoprecipitation (IP) was performed with the anti-Flag antibody and Western blots with the anti-Xpress antibody (top). The blot was stripped and rehybridized with anti-Flag antibody (bottom). Arrows indicate specific bands; (B) HDAC1-RARα heterodimerizes with RXRα within the cell. 293T cells were transfected as indicated. IP was performed with anti-Flag and immunoblot blot analysis was performed with an anti-RXRα antibody (top). The blots were stripped and rehybridized with anti-Flag antibody (bottom). Flag-RARα was used as a positive control. (C) HDAC1-RARα homodimers and HDAC1-RARα/RXRα bind to DR5 in vitro. (top) In vitro translated proteins were incubated with ³²P-labeled DR5 probe as indicated and resolved by electrophoresis. Competition and bandshift experiments were performed as indicated (bottom). Arrows indicate specific protein–DNA complexes. HR: HDAC1-RARα, hom: homodimer, het: heterodimer, *, supershifted band with anti-RXRα antibody; **, nonspecific bands.

Figure 3.

Biological properties of HDAC1-RARα. (A) HDAC1-RARα is a transcriptional repressor. Luciferase assay in transfected 293 cells. (black bars) RA-treated cells; (white bars) untreated cells. Luciferase activities were expressed relative to the value of lysates transfected with the reporter alone. (B) HDAC1-RARα binds to RARE and deacetylates histone H3. (top) ChIP assay on lysate of transiently transfected 293T cells with the indicated antibodies. PCR was performed with RARE specific primers. (middle) PCR analysis performed with RARE-specific primers on the cell lysates was used for ChIP assay. (bottom) The intensity of the bands was determined by densitometry. The value obtained from the lysate transfected with the empty vector is expressed as 1. (C) HDAC1-RARα represses the acetylation of histone H3 and H4 induced by BrHAT. 293T cells were cotransfected as indicated and the lysate was immunoblotted. The same membrane was hybridized and stripped in series with the indicated antibodies. The arrows indicate transfected Flag-tagged proteins (bottom). The ratio of acetylated/total histone levels was assessed by densitometry and provided by the histogram at the bottom. The value obtained from the lysate transfected with pCMV alone is expressed arbitrarily as 100%. The value for acetylated H3/total H3 and acetylated H4/total H4 is provided. (D) HDAC1-RARα inhibits the differentiation of U937. Cells were retrovirally transduced as indicated. Transduced cells were isolated by cell sorting and cultured with or without 2 ng/ml of TGFβ1 in addition to 500 ng/ml of vitamin D₃ for 96 h. Expression of CD11b was detected by flow cytometry. (black bars) percentage of treated cells expressing CD11b; (white bars) untreated cells. After treatment, CD11b expression is significantly reduced in PLZF-RARα (P = 0.01) and HDAC1-RARα (P = 0.02). Error bars indicate standard deviations.

Figure 4.

Characteristics of leukemias induced by PML-RARαM₄ and RARαE. (A) Schematic representation of transgenic lines and respective leukemia incidence. (B) Peripheral blood (PB) and bone marrow cells (BM) from representative leukemic RARαE, PML-RARαM₄, and PML-RARα transgenic mice stained with the Wright-Giemsa stain (×1,000). Note the presence of leukemic blasts in both BM and PB. (C) Flow cytometric analysis of BM cells from representative leukemic RARαE, PML-RARα M₄, and PML-RARα transgenic mice. Anti–Mac-1 and c-kit antibodies were used. (green line) Isotypic control. The percentages of positive cells are given in the respective histograms. (D) Leukemia induced by RARαE is resistant to RA treatment in vivo. Percentage of leukemic cells present in the PB of two RARαE transgenic mice (blue) and three nude mice (red) transplanted with leukemic cells obtained from RARαE transgenic mice. The horizontal axis indicates the length of treatment in days. Crosses indicate the time of death of each animal. RA had no impact on the percentage of leukemic cells present in the PB.