Acute promyelocytic leukemia (APL) is uniquely sensitive to treatment with all-trans retinoic acid (ATRA), which results in the expression of genes that induce the terminal granulocytic differentiation of the leukemic blasts. Here we report the identification of two ATRA responsive genes in APL cells, ID1 and ID2. These proteins act as antagonists of basic helix-loophelix (bHLH) transcription factors. ATRA induced a rapid increase in ID1 and ID2, both in the APL cell line NB4 as well as in primary patient cells. In addition, a strong downregulation of E2A was observed. E2A acts as a general heterodimerization partner for many bHLH proteins that are involved in differentiation control in various tissues. The simultaneous upregulation of ID1 and ID2, and the downregulation of E2A suggest a role for bHLH proteins in the induction of differentiation of APL cells following ATRA treatment. To test the relevance of this upregulation, ID1 and ID2 were overexpressed in NB4 cells. Overexpression inhibited proliferation and induced a G0/G1 accumulation. These results indicate that ID1 and ID2 are important retinoic acid responsive genes in APL, and suggest that the inhibition of specific bHLH transcription factor complexes may play a role in the therapeutic effect of ATRA in APL.

IntroductionAcute promyelocytic leukemia (APL) is characterized by an excess of immature promyelocytes in the bone marrow that fail to differentiate toward mature granulocytes. In approximately 98% of the cases, the retinoic acid receptor-␣ (RAR␣) gene is fused to the promyelocytic leukemia (PML) gene resulting in a PML-RAR␣ fusion protein. The PML-RAR␣ chimeric protein contains most of the PML sequence and a large part of RAR␣, including its DNAand nuclear hormone-binding domains. APL blasts can be forced to terminally differentiate using pharmacological doses of all-trans retinoic acid (ATRA). When treated with chemotherapy, APL patients can be cured in approximately 40% of the cases. The combination of ATRA with chemotherapy leads to a remarkably high cure rate of approximately 90%, 1,2 and APL currently represents the best prognostic group among the different forms of leukemia. This treatment constitutes one of the first examples of successful induction of differentiation of malignant cells yielding significant clinical results.The role of PML-RAR␣ in transformation and terminal differentiation has been studied intensively in the past decade. PML-RAR␣ was shown to act as a dominant oncogene, interfering with the normal function of the unrearranged PML as well as the unrearranged RAR␣ protein. Expression of the fusion protein in immature hematopoietic cells induced a maturation block at the promyelocytic stage. Inoculation of PML-RAR␣-transduced bone marrow cells into irradiated syngenic mice resulted in the development of retinoic acid-sensitive leukemia. 3,4 Furthermore, PML-RAR␣ transgenic mice developed a myeloproliferative syndrome that progressed to overt leukemia in 30% to 90% of the animals after 6 to 12 months, depending on the promoter that was used. [5][6][7] PML is a ubiquitously expressed protein that localizes to nuclear substructures termed nuclear bodies. More than 50 protein partners with various biologic functions colocalize with PML. 8 In APL cells, these nuclear bodies are disrupted and dispersed into numerous small microspeckles. 9 PML has multiple tumorsuppressor functions and is involved in growth control, replicative senescence, and apoptosis. 10 PML Ϫ/Ϫ mice are prone to develop tumors in response to various forms of stress. In addition, PML-RAR␣ transgenic mice develop leukemia much faster in a PML Ϫ/Ϫ background. 11 Retinoic acid receptors are transcription factors that activate genes in a ligand-dependent manner. RAR␣ binds to DNA as a heterodimer with RXR proteins. In the absence of ligand, both RAR␣/RXR and PML-RAR␣ bind corepressors such as N-Cor and SMRT and recruit histone deacetylases leading to gene silencing. In the presence of ligand, the corepressors are replaced by coactivators, leading to transcriptional activation. However, PML-RAR␣ releases the corepressors at much higher concentrations of ligand compared with the unrearranged receptors. Since PML-RAR␣ competes with unrearranged receptors for the same DNAbinding sites, the presence of the fusion protein results in dom...

PML-RARa is the product of the chromosomal translocation t(15;17) and is the causative oncogene in more than 98% of the cases of acute promyelocytic leukemia. PML-RARa interferes with the transcription of retinoic acid receptor target genes in a dominant manner, contributing to the transformation of the cells. RARa regulates transcription via heterodimerisation with RXR. PML-RARa can bind as a homodimer to the same sequences as RARa/RXR. At supra-physiological dosis of all-trans retionoic acid (ATRA), the block in differentiation in APL can be overcome. ATRA releases co-repressors from PML-RARa and activates RARa target genes. The ID1 gene is rapidly induced by ATRA in acute promyelocytic leukemia cells. Promoter analysis showed that the ID1 promoter was activated by PML-RARa but, unexpectedly, not by wild type RARa/RXR. Surprisingly, PML-RARa lacking the DNA binding domain could still transactivate the ID1 promoter indicating that transactivation was mediated without direct DNA binding. Promoter deletion studies showed that adjacent NF-Y and SP1 binding sites were essential for this transactivation. A direct physical interaction was shown between PML-RARa and SP1 in vitro and chromatin immunoprecepitation assays confirmed that a complex of PML-RARa/NF-Y/Sp1 is present on the ID1 promoter in vivo. In addition, we found that ectopic expression of PML-RARa induced expression of the endogenous ID1 gene in response to retinoic acid. We propose a novel, gain-of-function model for PML-RARa in which the fusion protein interferes with the transcription of SP1-NF-Y regulated genes. Interference is mediated without DNA-binding, through direct interaction with SP1. This implicates that apart from the previously described deregulation of retinoic acid receptor target genes, PML-RARa may deregulate an additional class of genes that are nomally not regulated by retinoid receptors.

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