Uveal melanoma is the most common intraocular tumor in adults, representing approximately 5% of all melanoma cases. Up to 50% of uveal melanoma patients develop metastases that are resistant to... Show moreUveal melanoma is the most common intraocular tumor in adults, representing approximately 5% of all melanoma cases. Up to 50% of uveal melanoma patients develop metastases that are resistant to most of the commonly used antineoplastic treatments. Virtually all uveal melanoma tumors harbor activating mutations in GNAQ or GNA11, encoding Gαq and Gα11, respectively. Constant activity of these proteins causes deregulation of multiple downstream signaling pathways including PKC, MAPK and YAP1/TAZ. While the importance of YAP1 signaling for the proliferation of uveal melanoma has recently been demonstrated, much less is known about the paralog of YAP1 transcriptional coactivator, named TAZ; however, similar to YAP1, TAZ is expected to be a therapeutic target in uveal melanoma. We performed a small-scale drug screen to discover a compound synergistically inhibiting uveal melanoma proliferation/survival in combination with YAP1/TAZ inhibition. We found that the combination of genetic depletion of YAP1/TAZ together with Mcl-1 inhibition demonstrates a synergistic inhibitory effect on the viability of uveal melanoma cell lines. Similarly, indirect attenuation of the YAP1/TAZ signaling pathway with an inhibitor of the mevalonate pathway, that is, the geranyl-geranyl transferase inhibitor GGTI-298, synergizes with Mcl-1 inhibition. This combination could be potentially used as a treatment for metastatic uveal melanoma. Show less
PURPOSE. Uveal melanoma (UM) is considered a rare disease; yet, it is the most common intraocular malignancy in adults. Although the primary tumor may be efficiently managed, more than 50% of... Show morePURPOSE. Uveal melanoma (UM) is considered a rare disease; yet, it is the most common intraocular malignancy in adults. Although the primary tumor may be efficiently managed, more than 50% of patients with UM develop distant metastases. The mortality at the first year after diagnosis of metastatic UM has been estimated at 81%, and the poor prognosis has not improved in the past years due to the lack of effective therapies. METHODS. In order to search for novel therapeutic possibilities for metastatic UM, we performed a small-scale screen of targeted drug combinations. We verified the targets of the tested compounds by western blotting and PCR and clarified the mechanism of action of the selected combinations by caspase 3 and 7 activity assay and flow cytometry. The best two combinations were tested in a mouse patient-derived xenograft (PDX) UM model as putative therapeutics for metastatic UM. RESULTS. Combinations of the multitarget drug trabectedin with either the CK2/CLK double-inhibitor CX-4945 (silmitasertib) or the c-MET/TAM (TYRO3, Axl, MERTK) receptor inhibitors foretinib and cabozantinib demonstrated synergistic effects and induced apoptosis (relative caspase 3 and 7 activity increased up to 20.5-fold in UM cell lines). In the case of the combination of foretinib and cabozantinib, inhibition of the TAM receptors, but not c-Met, was essential to inhibit the growth of UM cells. Monotreatment with trabectedin inhibited tumor growth by 42%, 49%, and 35% in the MM26, MM309, and MM339 PDX mouse models, respectively. CONCLUSIONS. Trabectedin alone or in combination with cabozantinib inhibited tumor growth in PDX UM mouse models. Blocking of MERTK, rather than TYRO3, activity inhibited UM cell growth and synergized with trabectedin. Show less
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... Show moreThe 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. Show less
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... Show moreSimple 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. Show less
Targeting the MAPK signaling pathway has transformed the treatment of metastatic melanoma. CRISPR-Cas9 genetic screens provide a genome-wide approach to uncover novel genetic dependencies that... Show moreTargeting the MAPK signaling pathway has transformed the treatment of metastatic melanoma. CRISPR-Cas9 genetic screens provide a genome-wide approach to uncover novel genetic dependencies that might serve as therapeutic targets. Here, we analyzed recently reported CRISPR-Cas9 screens comparing data from 28 melanoma cell lines and 313 cell lines of other tumor types in order to identify fitness genes related to melanoma. We found an average of 1,494 fitness genes in each melanoma cell line. We identified 33 genes, inactivation of which specifically reduced the fitness of melanoma. This set of tumor type-specific genes includes established melanoma fitness genes as well as many genes that have not previously been associated with melanoma growth. Several genes encode proteins that can be targeted using available inhibitors. We verified that genetic inactivation of DUSP4 and PPP2R2A reduces the proliferation of melanoma cells. DUSP4 encodes an inhibitor of ERK, suggesting that further activation of MAPK signaling activity through its loss is selectively deleterious to melanoma cells. Collectively, these data present a resource of genetic dependencies in melanoma that may be explored as potential therapeutic targets. Show less
Nejad, E.B.; Labrie, C.; Abdulrahman, Z.; Elsas, M.J. van; Rademaker, E.; Kleinovink, J.W.; ... ; Burg, S.H. van der 2020
Background Immunotherapy of cancer is successful but tumor regression often is incomplete and followed by escape. Understanding the mechanisms underlying this acquired resistance will aid the... Show moreBackground Immunotherapy of cancer is successful but tumor regression often is incomplete and followed by escape. Understanding the mechanisms underlying this acquired resistance will aid the development of more effective treatments. Methods We exploited a mouse model where tumor-specific therapeutic vaccination results in tumor regression, followed by local recurrence and resistance. In depth studies on systemic, local and tumor intrinsic changes were performed with flow and mass cytometry, immunohistochemistry, transcriptomics and several perturbation studies with inhibitors or agonistic antibodies in mice. Main findings were recapitulated in vaccinated patients. Results Full tumor regression and cure of tumor-bearing mice is dependent on the magnitude of the vaccine-induced T-cell response. Recurrence of tumors did not involve classical immune escape mechanisms, such as antigen-presentation alterations, immune checkpoint expression, resistance to killing or local immune suppression. However, the recurrent tumors displayed a changed transcriptome with alterations in p53, tumor necrosis factor-alpha and transforming growth factor-beta signaling pathways and they became immunologically cold. Remarkably, ex vivo cell-sorted recurrent tumors, directly reinjected in naive hosts retained their resistance to vaccination despite a strong infiltration with tumor-specific CD8(+)T cells, similar to that of vaccine-responsive tumors. The influx of inflammatory mature myeloid effector cells in the resistant tumors, however, was impaired and this turned out to be the underlying mechanisms as restoration of inflammatory myeloid cell infiltration reinstated the sensitivity of these refractory tumors to vaccination. Notably, impaired myeloid cell infiltration after vaccination was also associated with vaccine resistance in patients. Conclusion An immunotherapy-induced disability of tumor cells to attract innate myeloid effector cells formed a major mechanism underlying immune escape and acquired resistance. These data not only stresses the importance of myeloid effector cells during immunotherapy but also demands for new studies to harness their tumoricidal activities. Show less
The p53 tumor suppressor pathway is inactivated in cancer either via direct mutation or via deregulation of upstream regulators or downstream effectors. P53 mutations are rare in uveal melanoma.... Show moreThe p53 tumor suppressor pathway is inactivated in cancer either via direct mutation or via deregulation of upstream regulators or downstream effectors. P53 mutations are rare in uveal melanoma. Here we investigated the role of the p53 inhibitor Hdmx in uveal melanoma. We found Hdmx over-expression in a subset of uveal melanoma cell lines and fresh-frozen tumor samples. Hdmx depletion resulted in cell-line dependent growth inhibition, apparently correlating with differential Hdm2 levels. Surprisingly, p53 knockdown hardly rescued cell cycle arrest and apoptosis induction upon Hdmx knockdown, whereas it effectively prevented growth suppression induced by the potent p53 activator Nutlin-3. In addition, two compounds inhibiting Hdmx function or expression, SAH-p53-8 and XI-011, also elicited a growth inhibitory effect in a partly p53-independent manner. These findings suggest a novel, growth-promoting function of Hdmx that does not rely on its ability to inhibit p53. We provide evidence for a contribution of p27 protein induction to the observed p53-independent G1 arrest in response to Hdmx knockdown. In conclusion, our study establishes the importance of Hdmx as an oncogene in a subset of uveal melanomas and widens the spectrum of its function beyond p53 inhibition. Show less
