Novel treatments of uveal melanoma identified with a synthetic lethal CRISPR/Cas9 screen

Animal science

Modeling of Human Uveal Melanoma in Zebrafish Xenograft Embryos

Animal science

Targeting MDMX and PKC delta to improve current uveal melanoma therapeutic strategies

Cancer Signaling networks and Molecular Therapeutic

Novel Treatments of Uveal Melanoma Identified with a Synthetic Lethal CRISPR/Cas9 Screen

Simple Summary We performed a CRISPR-Cas9 synthetic lethality screen in order to identify molecular targets whose inhibition would synergistically enhance the effect of everolimus in uveal melanoma cells. IGF1R and PRKDC, among others, were identified as hits. We verified these hits effects genetically: we treated the uveal melanoma cell lines depleted of PRKDC or IGF1R with everolimus and, in case of IGF1R, observed a synergistic effect. Additionally, we found synergistic growth inhibition with the inhibitors targeting DNA-PKcs or IGF1R in combination with everolimus. Moreover, we investigated the combination of targeted inhibitors of DNA-PKcs and IGF1R with everolimus on uveal melanoma in an in vivo model. The dual DNA-PKcs/mTOR inhibitor CC-115 demonstrated activity in vivo. Currently, no systemic treatment is approved as the standard of care for metastatic uveal melanoma (UM). mTOR has been evaluated as a drug target in UM. However, one of the main limitations is dose reduction due to adverse effects. The combination of everolimus with another targeted agent would allow the reduction of the dose of a single drug, thus widening the therapeutic window. In our study, we aimed to identify a synergistic combination with everolimus in order to develop a novel treatment option for metastatic UM. We exploited CRISPR-Cas9 synthetic lethality screening technology to search for an efficient combination. IGF1R and PRKDC and several other genes were identified as hits in the screen. We investigated the effect of the combination of everolimus with the inhibitors targeting IGF1R and DNA-PKcs on the survival of UM cell lines. These combinations synergistically slowed down cell growth but did not induce apoptosis in UM cell lines. These combinations were tested on PDX UM in an in vivo model, but we could not detect tumor regression. However, we could find significant activity of the dual DNA-PKcs/mTOR inhibitor CC-115 on PDX UM in the in vivo model.Cancer Signaling networks and Molecular Therapeutic

Chk2 mediates RITA-induced apoptosis

Reactivation of the p53 tumor-suppressor protein by small molecules like Nutlin-3 and RITA (reactivation of p53 and induction of tumor cell apoptosis) is a promising strategy for cancer therapy. The molecular mechanisms involved in the responses to RITA remain enigmatic. Several groups reported the induction of a p53-dependent DNA damage response. Furthermore, the existence of a p53-dependent S-phase checkpoint has been suggested, involving the checkpoint kinase Chk1. We have recently shown synergistic induction of apoptosis by RITA in combination with Nutlin-3, and we observed concomitant Chk2 phosphorylation. Therefore, we investigated whether Chk2 contributes to the cellular responses to RITA. Strikingly, the induction of apoptosis seemed entirely Chk2 dependent. Transcriptional activity of p53 in response to RITA required the presence of Chk2. A partial rescue of apoptosis observed in Noxa knockdown cells emphasized the relevance of p53 transcriptional activity for RITA-induced apoptosis. In addition, we observed an early p53- and Chk2-dependent block of DNA replication upon RITA treatment. Replicating cells seemed more prone to entering RITA-induced apoptosis. Furthermore, the RITA-induced DNA damage response, which was not a secondary effect of apoptosis induction, was strongly attenuated in cells lacking p53 or Chk2. In conclusion, we identified Chk2 as an essential mediator of the cellular responses to RITA

Novel Treatments of Uveal Melanoma Identified with a Synthetic Lethal CRISPR/Cas9 Screen

Simple Summary We performed a CRISPR-Cas9 synthetic lethality screen in order to identify molecular targets whose inhibition would synergistically enhance the effect of everolimus in uveal melanoma cells. IGF1R and PRKDC, among others, were identified as hits. We verified these hits effects genetically: we treated the uveal melanoma cell lines depleted of PRKDC or IGF1R with everolimus and, in case of IGF1R, observed a synergistic effect. Additionally, we found synergistic growth inhibition with the inhibitors targeting DNA-PKcs or IGF1R in combination with everolimus. Moreover, we investigated the combination of targeted inhibitors of DNA-PKcs and IGF1R with everolimus on uveal melanoma in an in vivo model. The dual DNA-PKcs/mTOR inhibitor CC-115 demonstrated activity in vivo. Currently, no systemic treatment is approved as the standard of care for metastatic uveal melanoma (UM). mTOR has been evaluated as a drug target in UM. However, one of the main limitations is dose reduction due to adverse effects. The combination of everolimus with another targeted agent would allow the reduction of the dose of a single drug, thus widening the therapeutic window. In our study, we aimed to identify a synergistic combination with everolimus in order to develop a novel treatment option for metastatic UM. We exploited CRISPR-Cas9 synthetic lethality screening technology to search for an efficient combination. IGF1R and PRKDC and several other genes were identified as hits in the screen. We investigated the effect of the combination of everolimus with the inhibitors targeting IGF1R and DNA-PKcs on the survival of UM cell lines. These combinations synergistically slowed down cell growth but did not induce apoptosis in UM cell lines. These combinations were tested on PDX UM in an in vivo model, but we could not detect tumor regression. However, we could find significant activity of the dual DNA-PKcs/mTOR inhibitor CC-115 on PDX UM in the in vivo model

Role of Mdm4 in drug sensitivity of breast cancer cells

The p53 tumor suppressor protein is frequently mutated in human tumors. It is thought that the p53 pathway is indirectly impaired in the remaining tumors, for example by overexpression of its important regulators Mdm2 and Mdm4, making them attractive targets for the development of anti-cancer agents. Recent studies have suggested that Mdm4 levels determine the sensitivity of tumor cells for anti-cancer therapy. To investigate this possibility, we studied the drug sensitivity of several breast cancer cell lines containing wild-type p53, but expressing different Mdm4 levels. We show that endogenous Mdm4 levels can affect the sensitivity of breast cancer cells to anti-cancer agents, but in a cell line-dependent manner and depending on an intact apoptotic response. Furthermore, treatment with the non-genotoxic agent Nutlin-3 sensitizes cells for doxorubicin, showing that activation of p53 by targeting its regulators is an efficient strategy to decrease cell viability of breast cancer cells. These results confirm a function of Mdm4 in determining the efficacy of chemotherapeutic agents to induce apoptosis of cancer cells in a p53-dependent manner, although additional undetermined factors also influence the drug response. Targeting Mdm4 to sensitize tumor cells for chemotherapeutic drugs might be a strategy to effectively treat tumors harboring wild-type p53. Oncogene (2010) 29, 2415-2426; doi:10.1038/onc.2009.522; published online 8 February 2010Microscopic imaging and technolog

Wild-type p53 inhibits pro-invasive properties of TGF-beta 3 in breast cancer, in part through regulation of EPHB2, a new TGF-beta target gene

Cancer Signaling networks and Molecular Therapeutic

Synergistic growth inhibition based on small-molecule p53 activation as treatment for intraocular melanoma

Ophthalmic researchGene regulation and cell differentiatio

MDMX Regulates Transcriptional Activity of p53 and FOXO Proteins to Stimulate Proliferation of Melanoma Cells

The tumor suppressor protein p53 has an important role in cell-fate determination. In cancer cells, the activity of p53 is frequently repressed by high levels of MDMX and/or MDM2. MDM2 is a ubiquitin ligase whose activity results in ubiquitin- and proteasome-dependent p53 degradation, while MDMX inhibits p53-activated transcription by shielding the p53 transactivation domain. Interestingly, the oncogenic functions of MDMX appear to be more wide-spread than inhibition of p53. The present study aimed to elucidate the MDMX-controlled transcriptome. Therefore, we depleted MDMX with four distinct shRNAs from a high MDMX expressing uveal melanoma cell line and determined the effect on the transcriptome by RNAseq. Biological function analyses indicate the inhibition of the cell cycle regulatory genes and stimulation of cell death activating genes upon MDMX depletion. Although the inhibition of p53 activity clearly contributes to the transcription regulation controlled by MDMX, it appeared that the transcriptional regulation of multiple genes did not only rely on p53 expression. Analysis of gene regulatory networks indicated a role for Forkhead box (FOX) transcription factors. Depletion of FOXO proteins partly prevented the transcriptional changes upon MDMX depletion. Furthermore, depletion of FOXO proteins relatively diminished the growth inhibition upon MDMX knockdown, although the knockdown of the FOXO transcription factors also reduces cell growth. In conclusion, the p53-independent oncogenic functions of MDMX could be partially explained by its regulation of FOXO activity