A Novel Binding Surface on the Retinoblastoma Tumor Suppressor Protein
Investigating the role of the “E2F1-specific” binding domain of Retinoblastoma Protein
Background and Significance
Mutations in the RB pathway occur in nearly all cancers. pRB regulates gene expression through physical interaction with E2F/DP heterodimeric transcription factors and through the recruitment of chromatin remodelling factors. Early studies suggested hypophosporylated pRb uses a “general” E2F binding site to bind and inhibit E2Fs1-4. Upon G1-S phase transition, successive phosphorylation of pRb by cyclin-dependent kinases (CDKs) releases pRB from the heterodimeric transcription factors which activate expression of transcriptional programs implicated in numerous cellular activities, such as cell cycle progression, DNA damage response, apoptosis, and differentiation. Recent studies show hyperphosphorylated pRB (ppRB) binds E2F1 through a second “E2F1-specific” site at the pRB c-terminus. E2F1 induces apoptosis in response to DNA damage through p53 or p73-dependent mechanisms.
Hypothesis:
The “E2F1-specific” site found at the pRB c-terminus represents a distinct method for CDK-mediated hyperphosphorylated pRb to regulate E2F1 responsive genes and E2F1-mediated apoptosis beyond the G1 phase of cell cycle.
Aim 1: Develop a gene-targeted mouse model that selectively disrupts the “E2F1-specific” binding site (Rb1 ΔS) on pRB.
Chimeras carrying the targeted Rb1 allele are currently under analysis for germ-line transmission. This in vivo model will reveal the role for the “E2F1-specific” interaction in development and tumorigenesis. Cell culture experiments demonstrate this interaction to be dispensable for cell cycle control, but indispensible for control of E2F1-mediated apoptosis. E2f1-/- mice develop an enlarged thymus with elevated levels of immature T-cells, and are predisposed to a variety of cancers, most commonly lymphoma. Thymocytes from E2f1-/- mice are resistant to apoptotic stimuli. Rb1ΔS/ΔS mice may display opposite characteristics. Inability of pRB to attenuate E2F1-mediated apoptosis may result in an underdeveloped thymus, and compromised immune system. Mouse embryonic fibroblasts (MEFs) harvested from Rb1ΔS/ΔS mice will be used in functional assays to confirm the role of this interaction in cell cycle control vs. apoptosis. Rb1 ΔS/ΔS mice will be crossed with other gene targeted mice which disrupt the general E2F binding site of pRB to allow for study of pRB-E2F regulation in isolation, without disruption of other pathways that utilize pRB or E2Fs.
Aim 2: Develop a drug-screening system to identify small molecules capable of inhibiting pRB-E2F1 binding through the “E2F1-specific” binding site.
Initial efforts are underway to develop a bacterial system for co-expression and purification of recombinant E2F1-DP1 heterodimers which will be used in the FRET-based drug screen. Recombinant E2F1 mutants will also be used to identify E2F1 modifications that allow or inhibit the interaction. The screen will be utilized in collaboration with the Ontario Institute for Cancer Research (OICR) to identify small molecules. Candidate molecules can be used in several mouse models to evaluate whether increased concentrations of free E2F1 after release from the “specific site” on pRb may represent a viable therapeutic target.
Relevance to Cancer
Various cancers over-express cyclins to promote CDK-mediated hyperphosphorylation of pRb. This project seeks to understand how transformed cells may exploit constitutive ppRB-E2F1 binding to attenuate DNA-damage induced E2F1-mediated apoptosis while allowing de-regulated proliferation, and to investigate the therapeutic potential of disrupting this interaction.
Background and Significance
Mutations in the RB pathway occur in nearly all cancers. pRB regulates gene expression through physical interaction with E2F/DP heterodimeric transcription factors and through the recruitment of chromatin remodelling factors. Early studies suggested hypophosporylated pRb uses a “general” E2F binding site to bind and inhibit E2Fs1-4. Upon G1-S phase transition, successive phosphorylation of pRb by cyclin-dependent kinases (CDKs) releases pRB from the heterodimeric transcription factors which activate expression of transcriptional programs implicated in numerous cellular activities, such as cell cycle progression, DNA damage response, apoptosis, and differentiation. Recent studies show hyperphosphorylated pRB (ppRB) binds E2F1 through a second “E2F1-specific” site at the pRB c-terminus. E2F1 induces apoptosis in response to DNA damage through p53 or p73-dependent mechanisms.
Hypothesis:
The “E2F1-specific” site found at the pRB c-terminus represents a distinct method for CDK-mediated hyperphosphorylated pRb to regulate E2F1 responsive genes and E2F1-mediated apoptosis beyond the G1 phase of cell cycle.
Aim 1: Develop a gene-targeted mouse model that selectively disrupts the “E2F1-specific” binding site (Rb1 ΔS) on pRB.
Chimeras carrying the targeted Rb1 allele are currently under analysis for germ-line transmission. This in vivo model will reveal the role for the “E2F1-specific” interaction in development and tumorigenesis. Cell culture experiments demonstrate this interaction to be dispensable for cell cycle control, but indispensible for control of E2F1-mediated apoptosis. E2f1-/- mice develop an enlarged thymus with elevated levels of immature T-cells, and are predisposed to a variety of cancers, most commonly lymphoma. Thymocytes from E2f1-/- mice are resistant to apoptotic stimuli. Rb1ΔS/ΔS mice may display opposite characteristics. Inability of pRB to attenuate E2F1-mediated apoptosis may result in an underdeveloped thymus, and compromised immune system. Mouse embryonic fibroblasts (MEFs) harvested from Rb1ΔS/ΔS mice will be used in functional assays to confirm the role of this interaction in cell cycle control vs. apoptosis. Rb1 ΔS/ΔS mice will be crossed with other gene targeted mice which disrupt the general E2F binding site of pRB to allow for study of pRB-E2F regulation in isolation, without disruption of other pathways that utilize pRB or E2Fs.
Aim 2: Develop a drug-screening system to identify small molecules capable of inhibiting pRB-E2F1 binding through the “E2F1-specific” binding site.
Initial efforts are underway to develop a bacterial system for co-expression and purification of recombinant E2F1-DP1 heterodimers which will be used in the FRET-based drug screen. Recombinant E2F1 mutants will also be used to identify E2F1 modifications that allow or inhibit the interaction. The screen will be utilized in collaboration with the Ontario Institute for Cancer Research (OICR) to identify small molecules. Candidate molecules can be used in several mouse models to evaluate whether increased concentrations of free E2F1 after release from the “specific site” on pRb may represent a viable therapeutic target.
Relevance to Cancer
Various cancers over-express cyclins to promote CDK-mediated hyperphosphorylation of pRb. This project seeks to understand how transformed cells may exploit constitutive ppRB-E2F1 binding to attenuate DNA-damage induced E2F1-mediated apoptosis while allowing de-regulated proliferation, and to investigate the therapeutic potential of disrupting this interaction.