Lecture #14 | Fusion Proteins and Designer Drugs: PML-RAR & Retinoids BCR-ABL & Gleevec

How Proto-oncogenes Become Oncogenes

• Deletion or point mutation in coding sequence: Results in a hyperactive protein made in normal amounts.

• Gene amplification: Leads to normal protein greatly overproduced.

• Chromosome rearrangement caused by ionizing radiation:

  • Nearby regulatory DNA sequence causes normal protein to be overproduced.

  • Fusion to actively transcribed gene greatly overproduces fusion protein, or fusion protein is hyperactive.

Oncogenic Fusion Proteins Overview

• Product of chromosomal translocations.

• Contain new properties due to modular nature of protein domains.

• Frequently found in leukemias.

• Often involve:

  • Transcription factors (e.g., PML-RAR).

  • Signal transduction pathway (e.g., BCR-ABL).

• Can be targets for cancer therapy:

  • Retinoids target PML-RAR.

  • Gleevec targets BCR-ABL.

Translocations in Acute Leukemias

• Many translocations are present in various acute leukemias and involve oncogenes.

• Specific translocations are repeatedly found in cancers from different people.

  • Why leukemias?

    • Translocation in T cells

Translocation t(15;17), PML-RAR

• Reciprocal translocation between chromosome 15 & 17.

• Found in Acute Promyelocytic Leukemia (APL).

• Caused by bimolane, a topoisomerase II inhibitor used as a medical treatment in China.

• Fuses retinoic acid receptor (RARα) to PML.

• PML: zinc fingers and coiled-coil domains.

• RARα: DNA and ligand binding domains (DBD, LBD).

Retinoic Acid Receptor -- RAR

• ~460 amino acids.

• Retinoic acid (RA) studied for over a century.

• RARs studied since late 1980s.

• RA is formed from dietary Vitamin A in dairy products, fish liver oils, and β-carotene from plants.

• RA is essential for normal development and plays a critical role in differentiation, cell growth, and death.

• RA has tumor suppressive activities via its ability to promote differentiation and apoptosis.

• too much RA is teratogenic (causes birth defects).

• RAR is a member of a superfamily of ligand-dependent transcription factors (nuclear receptors, NR).

• RAR forms heterodimers with retinoid X receptor (RXR).

• Dimers bind retinoic acid response elements (RAREs).

• In the absence of RA, RAR binds co-repressors.

  • N-CoR (SMRT) forms a complex with Sin3A & HDAC.

• Upon ligand (RA) binding, RAR undergoes a conformational change.

  • Co-repressors are released.

  • Co-activators with HAT activity are recruited.

  • The Mediator complex binds and interacts directly with the basal transcription machinery which initiates transcription.

• Many target genes of RAR have been identified with roles in development, metabolism, differentiation, et

  • All have RARE

Nuclear Receptors domains

• DNA binding domain (DBD):

  • 2 zinc fingers, each consisting of 4 Cysteines.

• Ligand binding domain (LBD):

  • ligand binding pocket (LBP) – hydrophobic.

  • activation function (AF-2) – binds Co-activators (& Co-repressors).

• N-terminal activation function (AF-1):

  • ligand-independent, binds Co-activators, interactions with LBD.

    

Ligands for nuclear receptors

Unligated

RAR:RXR binds RARE and co-repressors with HDAC activity.

Ligated RAR

recruits co-activators with HAT activity and then Mediator complex (SMCC/TRAP/DRIP).

PML – Promyelocytic Leukemia Protein

• Identified in 1977 as part of the PML-RAR fusion protein, before RAR was cloned.

• PML contains 3 ring fingers, a zipper motif and a coiled-coil region that promotes oligomerization.

  • increase activity and stability

• Ability to oligomerize is critical to the oncogenic function of the fusion protein.

PML-RAR fusion protein function: Alters Expression of RA-Responsive Genes

• PML-RAR has the same DNA binding specificity as RAR.

  • Dimerization with RXR is not needed for PML-RAR to bind DNA.

  • PML provides a dimerization motif with its coiled coil region.

• PML-RAR has the same affinity for RA as wildtype RAR, but more RA is required to remove the co-repressor.

• Co-repressor with HDAC bound.

• Treatment with high doses of RA leads to Co-activator with HAT bound.

• PML-RAR blocks cells in pro-myelocytic (pre-differentiation) stage unless high doses of RA are given.

  • low = physiological concentrations

  • high = pharmacological/therapeutic concentrations → for leukemia cells

PML-RAR fusion protein function: Causes Nuclear Bodies to Disintegrate

• PML is found in nuclear bodies (NB), heterogeneous, dynamic macromolecular structures in the nucleus:

  • Sequester and release proteins

  • Mediate post-translational modification of proteins

  • Promote specific events in response to cellular stress

  • Associate with transcriptionally active regions of the genome

  • Role in apoptosis, senescence, tumor suppression, telomere length

• PML interacts with several proteins in NBs, including p53, an important tumor suppressor protein.

BCR-ABL Fusion Protein (overview)

• Translocation t(9;22) between c9 and c22 (Philadelphia chromosome).

• Present in 90% of CML (chronic myelogenous leukemia) and 10-20% ALL (acute lymphoblastic leukemia).

• Gleevec – designer drug

• BCR-ABL fusion protein has constitutive kinase activity and phosphorylates new substrates

BCR-ABL Activity

• stimulates proliferation via Ras/MAPK: interacts with SH2 of adaptor GRB2 → activates Ras → GF-independent cell growth

• activates Src: Increase motility Decrease adhesion

• suppresses apoptosis: activates PI3K pathway

• activates JAK/STAT: cytokine pathway

Gleevec

miracle drug

• very effective at treating CML

• highly specific with few side effects

• in the ATP binding pocket of the

  tyrosine kinase domain of BCR-ABL

  • Prevents ATP from binding and the kinase from transmitting the signal

  • New properties of Bcr-Abl make it a good anti-cancer target

    locks BCR-ABL in the inactive form

Downsides to Gleevac

Patients can develop resistance to Gleevac via mutations in the Gleevac binding site

• Does not work when a mutation has locked Abl into the active state

• Dasatinib, another Src kinase inhibitor, does bind the active form

• Inhibitors of Src family kinases are used to treat cancer of the lung, colon, stomach, and other leukemias