Project description:Two genes have a synthetic lethal relationship when silencing or inhibition of one gene is only lethal in the context of a mutation or activation of the second gene. This situation offers an attractive therapeutic strategy, as inhibition of such a gene will only trigger cell death in tumor cells with an activated second oncogene but spare normal cells without activation of the second oncogene. Here we present evidence that CDK2 is synthetic lethal to neuroblastoma cells with MYCN amplification and overexpression. Neuroblastomas are childhood tumors with an often lethal outcome. Twenty percent of the tumors have MYCN amplification and these tumors are ultimately refractory to any therapy. Targeted silencing of CDK2 by three RNA interference techniques induced apoptosis in MYCN-amplified neuroblastoma cell lines, but not in MYCN single copy cells. Silencing of MYCN abrogated this apoptotic response in MYCN-amplified cells. Inversely, silencing of CDK2 in MYCN single copy cells did not trigger apoptosis, unless a MYCN transgene was activated. The MYCN induced apoptosis after CDK2 silencing was accompanied by nuclear stabilization of P53 and mRNA profiling showed up-regulation of P53 target genes. Silencing of P53 rescued the cells from MYCN-driven apoptosis. The synthetic lethality of CDK2 silencing in MYCN activated neuroblastoma cells can also be triggered by inhibition of CDK2 with a small molecule drug. Treatment of neuroblastoma cells with Roscovitine, a CDK inhibitor, at clinically achievable concentrations induced MYCN-dependent apoptosis. The synthetic lethal relation between CDK2 and MYCN indicates CDK2 inhibitors as potential MYCN-selective cancer therapeutics. CDK2 shRNA in a tet repressor system was stably transfected in the IMR32 cell line. Time course analysis was performed in triplicate after induction of CDK2 shRNA at 5 time points.
Project description:Two genes have a synthetic lethal relationship when silencing or inhibition of one gene is only lethal in the context of a mutation or activation of the second gene. This situation offers an attractive therapeutic strategy, as inhibition of such a gene will only trigger cell death in tumor cells with an activated second oncogene but spare normal cells without activation of the second oncogene. Here we present evidence that CDK2 is synthetic lethal to neuroblastoma cells with MYCN amplification and overexpression. Neuroblastomas are childhood tumors with an often lethal outcome. Twenty percent of the tumors have MYCN amplification and these tumors are ultimately refractory to any therapy. Targeted silencing of CDK2 by three RNA interference techniques induced apoptosis in MYCN-amplified neuroblastoma cell lines, but not in MYCN single copy cells. Silencing of MYCN abrogated this apoptotic response in MYCN-amplified cells. Inversely, silencing of CDK2 in MYCN single copy cells did not trigger apoptosis, unless a MYCN transgene was activated. The MYCN induced apoptosis after CDK2 silencing was accompanied by nuclear stabilization of P53 and mRNA profiling showed up-regulation of P53 target genes. Silencing of P53 rescued the cells from MYCN-driven apoptosis. The synthetic lethality of CDK2 silencing in MYCN activated neuroblastoma cells can also be triggered by inhibition of CDK2 with a small molecule drug. Treatment of neuroblastoma cells with Roscovitine, a CDK inhibitor, at clinically achievable concentrations induced MYCN-dependent apoptosis. The synthetic lethal relation between CDK2 and MYCN indicates CDK2 inhibitors as potential MYCN-selective cancer therapeutics.
Project description:The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a paediatric cancer in which MYCN amplification is strongly associated with unfavourable outcome. Here, we show that CYC065, a clinical inhibitor of CDK2 and CDK9, selectively targets MYCN-amplified neuroblastoma via blockade of CDK9-dependent, MYCN-driven transcriptional elongation and CDK2-dependent proliferation. CYC065 also targets nascent transcription of short half-life genes including MYCN and MCL-1 leading to downregulation of MYCN-driven ‘adrenergic’ gene expression programs, growth inhibition, and apoptosis in vitro and in vivo. These data highlight the clinical potential of CDK2/9 inhibition in the treatment of MYCN-amplified neuroblastoma.
Project description:The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a paediatric cancer in which MYCN amplification is strongly associated with unfavourable outcome. Here, we show that CYC065, a clinical inhibitor of CDK2 and CDK9, selectively targets MYCN-amplified neuroblastoma via blockade of CDK9-dependent, MYCN-driven transcriptional elongation and CDK2-dependent proliferation. CYC065 also targets nascent transcription of short half-life genes including MYCN and MCL-1 leading to downregulation of MYCN-driven ‘adrenergic’ gene expression programs, growth inhibition, and apoptosis in vitro and in vivo. These data highlight the clinical potential of CDK2/9 inhibition in the treatment of MYCN-amplified neuroblastoma.
Project description:The most frequent focal alterations in human retinoblastoma are RB mutation and MYCN amplification. Whether MYCN overexpression drives retinoblastoma has not been assessed in model systems. Here, we show that Rb inactivation collaborates strongly with MYCN overexpression to lead to retinoblastoma in mice. MYCN overexpression in the context of Rb inactivation increased the expression of MYC, E2F and ribosome related gene sets, promoted excessive proliferation and led to retinoblastoma with anaplastic changes. Part of our study compares expression profiles in Rbnull vs Rbnull/MYCN overexpressing retinas collected at 12 days after birth.
Project description:sgRNA whole genome library sequencing in OCI-AML5-Cas9 EKO library cells overexpressing HMGA-YFP or control YFP vectors. The goal of this experiment is to identify synthetic lethal and synthetic rescue sgRNAs with regard to HMGA2 overexpression in AML.
Project description:Background: MYCN gene is a transcription factor whose amplification and overexpression can lead to tumorigenesis. In retina, MYCN overexpression is related to tumorigenesis of retinoblastoma, a childhood cancer. Purpose: To find out differentially expressed genes in MYCN-overexpressing cells, which will give information of MYCN-related gene expression and possibly find out new pathways in mechanism of retinoblastoma tumorigenesis with MYCN overexpression. Methods: MYCN-overexpressing cells were taken from MYCN electroporated E14 chicken retina and kept in culture for over 4 weeks. Unelectroporated E14 chicken retina central region was dissected as a control. Total RNA was extracted and sequenced on Illumina NovaSeq 6000. The data were used for differential expression analysis and GO/Pathway enrichment analysis. Results: Genes related to proliferation were upregulated in MYCN-overexpressing cells, while genes related to neural differentiation were downregulated. GO/Pathway enrichment analysis showed that upregulated genes were enriched for pathways associated with cell proliferation and downregulated genes for neural differentiation.
Project description:Background: MYCN gene is a transcription factor whose amplification and overexpression can lead to tumorigenesis. In retina, MYCN overexpression is related to tumorigenesis of retinoblastoma, a childhood cancer. Purpose: To find out differentially expressed genes in MYCN-overexpressing cells, which will give information of MYCN-related gene expression and possibly find out new pathways in mechanism of retinoblastoma tumorigenesis with MYCN overexpression. Methods: MYCN-overexpressing cells were taken from MYCN electroporated E14 chicken retina and kept in culture. Unelectroporated E14 chicken retina central region was dissected as a control. Total RNA was extracted and sequenced on Illumina NovaSeq 6000. The data were used for differential expression analysis and GO/Pathway enrichment analysis.
Project description:Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. Using a MYC target gene signature that predicts poor neuroblastoma prognosis we identified the histone chaperone, FAcilitates Chromatin Transcription (FACT), as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small molecule Curaxin compound, CBL0137, markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with chemotherapy in standard use by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN amplified neuroblastoma cells and a treatment strategy for MYCN-driven neuroblastoma