The Chromatin Remodeler CHD4 Sustains Ewing Sarcoma Cell Survival by Controlling Global Chromatin Architecture.

Ewing sarcoma is an aggressive cancer with a defective response to DNA damage leading to an enhanced sensitivity to genotoxic agents. Mechanistically, Ewing sarcoma is driven by the fusion transcription factor EWS-FLI1, which reprograms the tumor cell epigenome. The nucleosome remodeling and deacetylase (NuRD) complex is an important regulator of chromatin function, controlling both gene expression and DNA damage repair, and has been associated with EWS-FLI1 activity. Here, a NuRD-focused CRISPR/Cas9 inactivation screen identified the helicase CHD4 as essential for Ewing sarcoma cell proliferation. CHD4 silencing induced tumor cell death by apoptosis and abolished colony formation. Although CHD4 and NuRD colocalized with EWS-FLI1 at enhancers and super-enhancers, CHD4 promoted Ewing sarcoma cell survival not by modulating EWS-FLI1 activity and its oncogenic gene expression program but by regulating chromatin structure. CHD4 depletion led to a global increase in DNA accessibility and induction of spontaneous DNA damage, resulting in an increased susceptibility to DNA-damaging agents. CHD4 loss delayed tumor growth in vivo, increased overall survival, and combination with PARP inhibition by olaparib treatment further suppressed tumor growth. Collectively, these findings highlight the NuRD subunit CHD4 as a therapeutic target in Ewing sarcoma that can potentiate the antitumor activity of genotoxic agents. SIGNIFICANCE: CRISPR/Cas9 screening in Ewing sarcoma identifies a dependency on CHD4, which is crucial for the maintenance of chromatin architecture to suppress DNA damage and a promising therapeutic target for DNA damage repair-deficient malignancies.

CRISPR activation screen identifies TGFß-associated PEG10 as a crucial tumor suppressor in Ewing sarcoma.

As the second most common pediatric bone and soft tissue tumor, Ewing sarcoma (ES) is an aggressive disease with a pathognomonic chromosomal translocation t(11;22) resulting in expression of EWS-FLI1, an "undruggable" fusion protein acting as transcriptional modulator. EWS-FLI1 rewires the protein expression in cancer cells by activating and repressing a multitude of genes. The role and contribution of most repressed genes remains unknown to date. To address this, we established a CRISPR activation system in clonal SKNMC cell lines and interrogated a custom focused library covering 871 genes repressed by EWS-FLI1. Among the hits several members of the TGFß pathway were identified, where PEG10 emerged as prime candidate due to its strong antiproliferative effect. Mechanistic investigations revealed that PEG10 overexpression caused cellular dropout via induction of cell death. Furthermore, non-canonical TGFß pathways such as RAF/MEK/ERK, MKK/JNK, MKK/P38, known to lead to apoptosis or autophagy, were highly activated upon PEG10 overexpression. Our study sheds new light onto the contribution of TGFß signalling pathway repression to ES tumorigenesis and suggest that its re-activation might constitute a novel therapeutic strategy.

Inhibition of HDACs reduces Ewing sarcoma tumor growth through EWS-FLI1 protein destabilization.

Oncogenic transcription factors lacking enzymatic activity or targetable binding pockets are typically considered "undruggable". An example is provided by the EWS-FLI1 oncoprotein, whose continuous expression and activity as transcription factor are critically required for Ewing sarcoma tumor formation, maintenance, and proliferation. Because neither upstream nor downstream targets have so far disabled its oncogenic potential, we performed a high-throughput drug screen (HTS), enriched for FDA-approved drugs, coupled to a Global Protein Stability (GPS) approach to identify novel compounds capable to destabilize EWS-FLI1 protein by enhancing its degradation through the ubiquitin-proteasome system. The protein stability screen revealed the dual histone deacetylase (HDAC) and phosphatidylinositol-3-kinase (PI3K) inhibitor called fimepinostat (CUDC-907) as top candidate to modulate EWS-FLI1 stability. Fimepinostat strongly reduced EWS-FLI1 protein abundance, reduced viability of several Ewing sarcoma cell lines and PDX-derived primary cells and delayed tumor growth in a xenograft mouse model, whereas it did not significantly affect healthy cells. Mechanistically, we demonstrated that EWS-FLI1 protein levels were mainly regulated by fimepinostat's HDAC activity. Our study demonstrates that HTS combined to GPS is a reliable approach to identify drug candidates able to modulate stability of EWS-FLI1 and lays new ground for the development of novel therapeutic strategies aimed to reduce Ewing sarcoma tumor progression.

Paracrine Placental Growth Factor Signaling in Response to Ionizing Radiation Is p53-Dependent and Contributes to Radioresistance.

Placental growth factor (PlGF) is a pro-angiogenic, N-glycosylated growth factor, which is secreted under pathologic situations. Here, we investigated the regulation of PlGF in response to ionizing radiation (IR) and its role for tumor angiogenesis and radiosensitivity. Secretion and expression of PlGF was induced in multiple tumor cell lines (medulloblastoma, colon and lung adenocarcinoma) in response to irradiation in a dose- and time-dependent manner. Early upregulation of PlGF expression and secretion in response to irradiation was primarily observed in p53 wild-type tumor cells, whereas tumor cells with mutated p53 only showed a minimal or delayed response. Mechanistic investigations with genetic and pharmacologic targeting of p53 corroborated regulation of PlGF by the tumor suppressor p53 in response to irradiation under normoxic and hypoxic conditions, but with so far unresolved mechanisms relevant for its minimal and delayed expression in tumor cells with a p53-mutated genetic background. Probing a paracrine role of IR-induced PlGF secretion in vitro, migration of endothelial cells was specifically increased towards irradiated PlGF wild type but not towards irradiated PlGF-knockout (PIGF-ko) medulloblastoma cells. Tumors derived from these PlGF-ko cells displayed a reduced growth rate, but similar tumor vasculature formation as in their wild-type counterparts. Interestingly though, high-dose irradiation strongly reduced microvessel density with a concomitant high rate of complete tumor regression only in the PlGF-ko tumors. IMPLICATIONS: Our study shows a strong paracrine vasculature-protective role of PlGF as part of a p53-regulated IR-induced resistance mechanism and suggest PlGF as a promising target for a combined treatment modality with RT.

YAP/TAZ inhibition reduces metastatic potential of Ewing sarcoma cells.

Ewing sarcoma (EwS) is a highly metastatic bone cancer characterized by the ETS fusion oncoprotein EWS-FLI1. EwS cells are phenotypically highly plastic and switch between functionally distinct cell states dependent on EWS-FLI1 fluctuations. Whereas EWS-FLI1high cells proliferate, EWS-FLI1low cells are migratory and invasive. Recently, we reported activation of MRTFB and TEAD, effectors of RhoA and Hippo signalling, upon low EWS-FLI1, orchestrating key steps of the EwS migratory gene expression program. TEAD and its co-activators YAP and TAZ are commonly overexpressed in cancer, providing attractive therapeutic targets. We find TAZ levels to increase in the migratory EWS-FLI1low state and to associate with adverse prognosis in EwS patients. We tested the effects of the potent YAP/TAZ/TEAD complex inhibitor verteporfin on EwS cell migration in vitro and on metastasis in vivo. Verteporfin suppressed expression of EWS-FLI1 regulated cytoskeletal genes involved in actin signalling to the extracellular matrix, effectively blocked F-actin and focal-adhesion assembly and inhibited EwS cell migration at submicromolar concentrations. In a mouse EwS xenograft model, verteporfin treatment reduced relapses at the surgical site and delayed lung metastasis. These data suggest that YAP/TAZ pathway inhibition may prevent EwS cell dissemination and metastasis, justifying further preclinical development of YAP/TAZ inhibitors for EwS treatment.

USP19 deubiquitinates EWS-FLI1 to regulate Ewing sarcoma growth.

Ewing sarcoma is the second most common pediatric bone and soft tissue tumor presenting with an aggressive behavior and prevalence to metastasize. The diagnostic translocation t(22;11)(q24;12) leads to expression of the chimeric oncoprotein EWS-FLI1 which is uniquely expressed in all tumor cells and maintains their survival. Constant EWS-FLI1 protein turnover is regulated by the ubiquitin proteasome system. Here, we now identified ubiquitin specific protease 19 (USP19) as a regulator of EWS-FLI1 stability using an siRNA based screening approach. Depletion of USP19 resulted in diminished EWS-FLI1 protein levels and, vice versa, upregulation of active USP19 stabilized the fusion protein. Importantly, stabilization appears to be specific for the fusion protein as it could not be observed neither for EWSR1 nor for FLI1 wild type proteins even though USP19 binds to the N-terminal EWS region to regulate deubiquitination of both EWS-FLI1 and EWSR1. Further, stable shUSP19 depletion resulted in decreased cell growth and diminished colony forming capacity in vitro, and significantly delayed tumor growth in vivo. Our findings not only provide novel insights into the importance of the N-terminal EWSR1 domain for regulation of fusion protein stability, but also indicate that inhibition of deubiquitinating enzyme(s) might constitute a novel therapeutic strategy in treatment of Ewing sarcoma.