Retinoic Acid Receptor- α (RAR α ) & its role in Acute Promyelocytic Leukemia (APL)

1. Retinoic Acid Receptor-α (RARα) & its role in Acute Promyelocytic Leukemia (APL) Danielle Matsushima

2. Vitamin A to Retinoic Acid (RA) • Vitamin A is important for many biological processes: transcription, immune function, vision, embryonic development (axis patterning), skin health etc. • Vitamin A is absorbed by the body from food in the form called retinol. • Retinol is converted to RA through a series of enzymatic steps. • RA is important for gene transcription and interacts with specific nuclear receptors to initiate the transcriptional process.

3. Converting Retinol to Retinoic Acid • Retinol (Vitamin A) is the precursor form of RA • Retinol is bound to retinoid-binding protein-4 (RBP4) in the blood and enters the cell through the STRA6 membrane receptor • Retinol is converted to retinaldehyde (retinal) by retinol dehydrogenases or alcohol dehydrogenases • retinaldyhde is converted to RA by retinaldyhyde dehydrogenases (RALDHs) • There are two types of RAs: • all-trans-RA (ATRA) • 9-cis-RA • RA binds to cellular retinoic acid-binding proteins (CRABPs) which carry RA into the nucleus where it can then bind to receptors Maden, 2007.

4. Retinoic Acid Receptors • There are two types of receptors that RA interact with: • Retinoic Acid Receptors (RARs) • Retinoid X Receptors (RXRs) • Both RARs and RXRs have three forms: α, β, & γ • RARs act in combination with RXRs to form heterodimers • The RXR/RAR heterodimers act as ligand dependent transcriptional regulators by binding to RARE (retinoic acid response element) DNA sequences in the nucleus and initiate transcription if they are bound by RA Bastien & Rochette-Egly 2004 Maden 2007

5. Repressing Gene Transcription • When the RAR/RXR heterodimer is unliganded and bound to a RARE then the complex acts to repress transcription • Recruits corepressors NCoR and SMRT that interact with the lipid binding domains (LBDs) of both receptors • These corepressors also interact with the N-terminal histone tails of the nucleosomal DNA through histone deacetylase activity compelexes (HDACs) Bastien & Rochette-Egly 2004

6. Gene Transcription • When a ligand (RA) is attached to a RAR/RXR heterodimer then there is a conformational change in the LBD. • The conformational change causes higher DNA affinity to the RARE as well as corepressor release. • Then mediator complexes are recruited to induce gene transcription. Bastien & Rochette-Egly 2004

7. RAR/RXR heterodimers are involved in many pathways - RAR/RXR- mediated transcription can be controlled through ubiqutin-proteasome systems, phosphorylation and RAR/RXR heterodimers are involved in other signaling pathways.

8. Acute Promyelocytic Leukemia (APL) • Acute Promyelocytic Leukemia is a variant of Acute Myeloid Leukemia, specifically subtype M3. • Patients with APL suffer from an accumulation of immature granulocytes or promyelocytes in their blood and bone marrow. http://upload.wikimedia.org/wikipedia/commons/2/20/Illu_blood_cell_lineage.jpg

9. The cause of APL • 98% of APL patients have a reciprocal translocation between chromosomes 15 and 17. • The RARα gene is located on chromosome 17 and the promyelocytic leukemia (PML) gene is on chromosome 15. This translocation causes a fusion between the two genes which creates a fusion oncoprotein; PML-RARαaswell as its reversal, RARα-PML. • There are 4 other known translocations associated with APL and the RARα: the promyelocytic leukemia zinc finger gene (PLZF), the nucleophosmin gene (NPM), the nuclear mitotic apparatus gene (NuMA), and the signal transducer and activator of transcription 5b gene (Stat5b) Zelent et al. 2001

10. Effects of Fusion Proteins • Fusion proteins lead to transcriptional repression. PML-RARα can homodimerize as PML-RARα/ PML-RARα or heterodimerize as PML-RARα/RXR and either one interacts with SMRT and NCoR to recruit HDACs in the absence of pharmacological ATRA. • The fusion protein complexes are not activated by physiological concentrations of RA so the corepressors are never disassociated and transcription never occurs. • Gene transcription is essential for the differentiation of the promyelocytes to granulocytes but with PML-RARα instead of normal RARα and physiological ATRA levels, gene transcription does not take place. Zelent et al. 2001

11. RA and Clinical Remission • Only patients with the PML-RARα fusion protein respond to pharmacological doses of RA. The normal concentration of ATRA is 10-9 mol/l and levels of 10-6 mol/l are needed to offset effects of the abnormal PML-RARα complex. • The high dosage of RA is enough to bind to the PML-RARα fusion protein and dissociate the SMRT/HDAC complex so that repression is no longer occurs. Another potential mechanism is that high levels of RA free the RXR from the PML-RARα/RXR heterodimer so that RXR can then dimerize with wildtype RARα and restore normal transcriptional activity. Lin et al. 2001

12. Knockout Mice • RARα -/- mice show no obvious defects and are viable. This is true for the other RAR subtypes as well which suggests functional redundancy between the genes. However, when all three RAR variants are knocked out there is fetal lethality. Knockout mice also appear to have normal granulocyte levels in their peripheral blood and organs but RARα knockouts might control exogenous RA on myeloid cells. • The repression of myeloid differentiation can occur due to the fusion proteins blocking RA-dependent and RA-independent pathways. Lin et al. 2001 - Blockage can occur in RA-independent pathways when fusion proteins bind to targets that do not have typical RARE sequences and either activating or inhibiting their target genes (genes involved in the differentiation of promyelocytes)

13. To summarize… • Retinoic Acid is a Vitamin A derivative that acts as a ligand on RAR/RXR heterodimers. • When ligand bound, the heterodimers bind to DNA sequences called RAREs and initiate gene transcription through the disassociation of corepressor molecules on the DNA and recruitment of transcriptional activators. • Most APL cases are due to a reciprocal translocation between chromosomes 15 and 17 that fuse the RARα gene to the PML gene. This abnormal protein complex blocks differentiation of promyelocytes so there is an accumulation of immature cells in the blood and bone marrow leading to cancer. • Pharmacological levels of ATRA can induce normal differentiation of these cancerous cells.

14. Sources • http://www.dietandfitnesstoday.com/vitaminA.php • http://www.fotosearch.com/UPC005/rwl03011/ • http://www.med-med.virginia.edu/courses/path/innes/wcd/myeloid1.cfm • Bastien J and Rochette-Egly C. (2004). Nuclear retinoid receptors and the transcription of retinoid-target genes. Gene. 328, 1-16. • Germain P, Chambon P, Eichele G, Evans RM, Lazar MA, Leid M, De Lera AR, Lotan R, Mangelsdorf DJ, and Gronemeyer H. (2006). International Union of Pharmacology. LX. Retinoic Acid Receptors. Pharm. Rev.58, 712-725. • Kastner P and Chan S. (2001). Function of RARα during the maturation of neutrophils. Oncogene. 20, 7178-7185. • Lin RJ, Sternsdorf T, Tini M, and Evans RM. (2001). Transcriptional regulation in acute promyelocytic leukemia. Oncogene, 20, 7204-7215. • Maden M. (2007). Retinoic acid in the development, regeneration and maintenance of the nervous system. Nat. Neuro. 8, 755-765. • Sirulnik A, Melnick A, Zelent A, and Licht JD. (2003). Molecular pathogenesis of acute promyelocytic leukaemia and APL variants. BP & Res. Clin. Heam. 3, 387-408. • Zelent A, Buidez F, Melnick A, Waxman S, and Licht JD. (2001). Translocations of the RARα gene in acute promyelocytic leukemia. Oncogene. 20, 7186-7203.