Retinoic Acid Receptor-Retinoic Acid Receptor-αα (RAR(RARαα) & its role in Acute ) & its role in Acute

Promyelocytic Leukemia (APL)Promyelocytic Leukemia (APL)

Danielle MatsushimaDanielle Matsushima

Vitamin A to Retinoic Acid (RA)Vitamin A to Retinoic Acid (RA)

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

Converting Retinol to Retinoic AcidConverting 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.

Retinoic Acid ReceptorsRetinoic Acid ReceptorsThere are two types of receptors There are two types of receptors that RA interact with:that RA interact with:– Retinoic Acid Receptors (RARs)Retinoic Acid Receptors (RARs)

– Retinoid X Receptors (RXRs)Retinoid X Receptors (RXRs)

Both RARs and RXRs have three Both RARs and RXRs have three forms: forms: αα, , ββ, & , & γγRARs act in combination with RARs act in combination with RXRs to form heterodimers RXRs to form heterodimers The RXR/RAR heterodimers act as The RXR/RAR heterodimers act as ligand dependent transcriptional ligand dependent transcriptional regulators by binding to RARE regulators by binding to RARE (retinoic acid response element) (retinoic acid response element) DNA sequences in the nucleus DNA sequences in the nucleus and initiate transcription if they are and initiate transcription if they are bound by RAbound by RA

Bastien & Rochette-Egly 2004

Maden 2007

Repressing Gene TranscriptionRepressing Gene Transcription

When the RAR/RXR heterodimer is unliganded and When the RAR/RXR heterodimer is unliganded and bound to a RARE then the complex acts to repress bound to a RARE then the complex acts to repress transcriptiontranscription– Recruits corepressors NCoR and SMRT that interact with the Recruits corepressors NCoR and SMRT that interact with the

lipid binding domains (LBDs) of both receptorslipid binding domains (LBDs) of both receptors– These corepressors also interact with the N-terminal histone These corepressors also interact with the N-terminal histone

tails of the nucleosomal DNA through histone deacetylase tails of the nucleosomal DNA through histone deacetylase activity compelexes (HDACs) activity compelexes (HDACs)

Bastien & Rochette-Egly 2004

Gene TranscriptionGene Transcription

When a ligand (RA) is When a ligand (RA) is attached to a RAR/RXR attached to a RAR/RXR heterodimer then there is heterodimer then there is a conformational change a conformational change in the LBD.in the LBD.– The conformational change The conformational change

causes higher DNA affinity causes higher DNA affinity to the RARE as well as to the RARE as well as corepressor release.corepressor release.

– Then mediator complexes Then mediator complexes are recruited to induce are recruited to induce gene transcription.gene transcription.

Bastien & Rochette-Egly 2004

RAR/RXR heterodimers are RAR/RXR heterodimers are involved in many pathwaysinvolved 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.

Acute Promyelocytic Leukemia Acute Promyelocytic Leukemia (APL)(APL)

Acute Promyelocytic Acute Promyelocytic Leukemia is a variant of Leukemia is a variant of Acute Myeloid Leukemia, Acute Myeloid Leukemia, specifically subtype M3.specifically subtype M3.

Patients with APL suffer Patients with APL suffer from an accumulation of from an accumulation of immature granulocytes or immature granulocytes or promyelocytes in their promyelocytes in their blood and bone marrow. blood and bone marrow.

http://upload.wikimedia.org/wikipedia/commons/2/20/Illu_blood_cell_lineage.jpg

The cause of APLThe cause of APL98% of APL patients have a reciprocal translocation between 98% of APL patients have a reciprocal translocation between chromosomeschromosomes 15 and 17.15 and 17.

- The The RARRARαα gene is located on chromosome 17 and the gene is located on chromosome 17 and the promyelocytic promyelocytic leukemia (PML)leukemia (PML) gene is on chromosome 15. This translocation causes a gene is on chromosome 15. This translocation causes a fusion between the two genes which creates a fusion oncoprotein; PML-fusion between the two genes which creates a fusion oncoprotein; PML-RARRARαα asas well aswell as its reversal, RAR its reversal, RARαα-PML.-PML.

- There are 4 other known translocations associated with APL and the There are 4 other known translocations associated with APL and the RARRARαα: the : the promyelocytic leukemia zinc finger gene (PLZF),promyelocytic leukemia zinc finger gene (PLZF), the the nucleophosmin gene (NPM),nucleophosmin gene (NPM), the the nuclear mitotic apparatus gene (NuMA),nuclear mitotic apparatus gene (NuMA), and the and the signal transducer and activator of transcription 5b gene (Stat5b)signal transducer and activator of transcription 5b gene (Stat5b)

Zelent et al. 2001

Effects of Fusion ProteinsEffects of Fusion Proteins

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

Zelent et al. 2001

RA and Clinical RemissionRA and Clinical Remission

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

Lin et al. 2001

Knockout MiceKnockout MiceRARRARαα -/- mice show no obvious -/- mice show no obvious defects and are viable. This is defects and are viable. This is true for the other RAR subtypes true for the other RAR subtypes as well which suggests as well which suggests functional redundancy between functional redundancy between the genes. However, when all the genes. However, when all three RAR variants are knocked three RAR variants are knocked out there is fetal lethality. out there is fetal lethality. Knockout mice also appear to Knockout mice also appear to have normal granulocyte levels have normal granulocyte levels in their peripheral blood and in their peripheral blood and organs but RARorgans but RARαα knockouts knockouts might control exogenous RA on might control exogenous RA on myeloid cells.myeloid cells.The repression of myeloid The repression of myeloid differentiation can occur due to differentiation can occur due to the fusion proteins blocking RA-the fusion proteins blocking RA-dependent and RA-independent dependent and RA-independent pathways.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)

To summarize…To summarize…

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

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