Final Report Summary - DDR-MYCN-NB (The DNA damage response pathway in MYCN amplified neuroblastoma)
Background:
The MYC family of transcription factors are responsible for promoting cell cycle progression and their activity is frequently deregulated in cancer. Attempts to target MYC therapeutically have been problematic due to the fact that elevated MYC not only increases cell proliferation, but also and sensitises cells to apoptosis. Therefore therapeutic strategies must inhibit cell cycle progression without protecting cancer cells from apoptosis. In order to successfully target MYC it is necessary to understand precisely how MYC influences cell cycle checkpoints.
Objectives:
The main objective of this project was to create time resolved experimental data to mathematically model the effect of deregulated MYC expression on the G1 checkpoint in cancer cells. This will enable us to predict cell cycle and apoptosis outcomes after treatment with combinations of inhibitors against MYC, cell cycle components and DNA damaging agents.
Description of work done: Our model for deregulated MYC activity is based on neuroblastoma. 25% of neuroblastoma cases have MYCN amplification, where cells have 5-500 copies of the gene. MYCN amplification in neuroblastoma is a strong indicator for poor prognosis. To generate experimental data for the model we have used a MYCN amplified neuroblastoma cell line, in which we can reduce MYCN expression levels with a tetracycline inducible small hairpin RNA. The first step was to develop a synchronisation protocol for the cells, so that the whole cell population progressed through the cell cycle at the same time. The total length and phases of the cell cycle were carefully characterised in the presence and absence of MYCN (MYCN+/ MYCN-) before the levels of transcript and protein expression were profiled using RNA-sequencing and reverse phase protein arrays. This has provided a time resolved view of expression changes during each phase of the cell cycle. We designed RNA-sequencing experiments in order to take into account the changes in total message RNA levels during the cell cycle, by adding a spike-in control according to the precise number of cells in each sample. E2F is a transcription factor that governs the G1:S transition, and in order to investigate its activity through the cell cycle, an eGFP-E2F reporter construct was used in the synchronised IMR5/75 cells.
Main results:
IMR5/75 cells were synchronised at G1:S using a thymidine block. After thymidine was removed MYCN+ cells progressed synchronously through the cell cycle. MYCN- cells progressed through S-phase and G2 at a similar rate to MYCN+ cells, entering a non-cycling state after mitosis. The non-cycling state was defined by cells having a G1 complement of DNA, low levels of protein expression and low E2F activity. This result was unexpected, as MYC proteins are classically thought to affect the transition between G1 and S-phase. Expression profiling revealed that positive cell cycle regulators (e.g. CDK4 and CDK6) are up-regulated and negative regulators (e.g. CDKN1C and CDKN2D) are repressed in MYCN+ cells throughout the cell cycle. E2F transcript expression is induced at the G1:S transition, as expected, however we observe that E2F target gene expression is elevated in MYCN+ cells at during G2/M phase. Additionally, we observed that phosphorylation and deactivation of the retinoblastoma protein, a negative regulator of E2F occurs maximally during G2 /M phase. The data supports the novel hypothesis that the influence of MYC is during G2 phase, where it up-regulates CDK4 activity inactivating the retinoblastoma protein, preventing the cell from entering a non-cycling state. Analysis of our RNA-sequencing data has revealed that cell cycle phase has a strong impact on the global transcript level and it is necessary to take cell cycle phase into account during the analysis of gene expression changes.
Conclusions:
De-regulated MYC expression influences checkpoint decisions during G2 to determine cell cycle fate of daughter cells by preventing cells from entering a non-cycling state. Cell cycle phase has a profound effect on global transcription levels, indicating that MYC is not a transcriptional amplifier, but the effects of MYC are due to the different proportions of cycling and non-cycling cells in a population.
Potential impact:
Classically, MYC is thought to influence the G1: S checkpoint to promote cell proliferation. Data generated from this project suggests that de-regulated MYC affects cell cycle components during G2 of the cell cycle to influence entry into a non-cycling state, and not the G1:S transition. These findings impact greatly on our understanding of cell cycle control.
Socio-economic impact:
The model generated from this work will eventually be used to predict the best combinations of targeted therapies for cancers with deregulated MYC.
The MYC family of transcription factors are responsible for promoting cell cycle progression and their activity is frequently deregulated in cancer. Attempts to target MYC therapeutically have been problematic due to the fact that elevated MYC not only increases cell proliferation, but also and sensitises cells to apoptosis. Therefore therapeutic strategies must inhibit cell cycle progression without protecting cancer cells from apoptosis. In order to successfully target MYC it is necessary to understand precisely how MYC influences cell cycle checkpoints.
Objectives:
The main objective of this project was to create time resolved experimental data to mathematically model the effect of deregulated MYC expression on the G1 checkpoint in cancer cells. This will enable us to predict cell cycle and apoptosis outcomes after treatment with combinations of inhibitors against MYC, cell cycle components and DNA damaging agents.
Description of work done: Our model for deregulated MYC activity is based on neuroblastoma. 25% of neuroblastoma cases have MYCN amplification, where cells have 5-500 copies of the gene. MYCN amplification in neuroblastoma is a strong indicator for poor prognosis. To generate experimental data for the model we have used a MYCN amplified neuroblastoma cell line, in which we can reduce MYCN expression levels with a tetracycline inducible small hairpin RNA. The first step was to develop a synchronisation protocol for the cells, so that the whole cell population progressed through the cell cycle at the same time. The total length and phases of the cell cycle were carefully characterised in the presence and absence of MYCN (MYCN+/ MYCN-) before the levels of transcript and protein expression were profiled using RNA-sequencing and reverse phase protein arrays. This has provided a time resolved view of expression changes during each phase of the cell cycle. We designed RNA-sequencing experiments in order to take into account the changes in total message RNA levels during the cell cycle, by adding a spike-in control according to the precise number of cells in each sample. E2F is a transcription factor that governs the G1:S transition, and in order to investigate its activity through the cell cycle, an eGFP-E2F reporter construct was used in the synchronised IMR5/75 cells.
Main results:
IMR5/75 cells were synchronised at G1:S using a thymidine block. After thymidine was removed MYCN+ cells progressed synchronously through the cell cycle. MYCN- cells progressed through S-phase and G2 at a similar rate to MYCN+ cells, entering a non-cycling state after mitosis. The non-cycling state was defined by cells having a G1 complement of DNA, low levels of protein expression and low E2F activity. This result was unexpected, as MYC proteins are classically thought to affect the transition between G1 and S-phase. Expression profiling revealed that positive cell cycle regulators (e.g. CDK4 and CDK6) are up-regulated and negative regulators (e.g. CDKN1C and CDKN2D) are repressed in MYCN+ cells throughout the cell cycle. E2F transcript expression is induced at the G1:S transition, as expected, however we observe that E2F target gene expression is elevated in MYCN+ cells at during G2/M phase. Additionally, we observed that phosphorylation and deactivation of the retinoblastoma protein, a negative regulator of E2F occurs maximally during G2 /M phase. The data supports the novel hypothesis that the influence of MYC is during G2 phase, where it up-regulates CDK4 activity inactivating the retinoblastoma protein, preventing the cell from entering a non-cycling state. Analysis of our RNA-sequencing data has revealed that cell cycle phase has a strong impact on the global transcript level and it is necessary to take cell cycle phase into account during the analysis of gene expression changes.
Conclusions:
De-regulated MYC expression influences checkpoint decisions during G2 to determine cell cycle fate of daughter cells by preventing cells from entering a non-cycling state. Cell cycle phase has a profound effect on global transcription levels, indicating that MYC is not a transcriptional amplifier, but the effects of MYC are due to the different proportions of cycling and non-cycling cells in a population.
Potential impact:
Classically, MYC is thought to influence the G1: S checkpoint to promote cell proliferation. Data generated from this project suggests that de-regulated MYC affects cell cycle components during G2 of the cell cycle to influence entry into a non-cycling state, and not the G1:S transition. These findings impact greatly on our understanding of cell cycle control.
Socio-economic impact:
The model generated from this work will eventually be used to predict the best combinations of targeted therapies for cancers with deregulated MYC.