Therapeutic efficacy evaluation of agents that regulate epigenetic control of gene expression in bone sarcomas

L'identificazione di nuove strategie terapeutiche contro i sarcomi ossei rappresenta un obiettivo primario per il trattamento dei pazienti refrattari ai comuni agenti chemioterapici. Sia l’osteosarcoma (OS) che il sarcoma di Ewing (EWS), due fra i più comuni tumori ossei primari, derivano dalla trasformazione di cellule staminali mesenchimali (MSC) e/o progenitori osteoblastici in qualsiasi fase del differenziamento. La perdita del differenziamento è una caratteristica biologica specifica di questi tumori ed ha un forte significato prognostico. Ripristinare il differenziamento attraverso la reversione di alterazioni a livello epigenetico può rappresentare quindi un'importante opportunità terapeutica. Qui sono stati analizzati gli effetti su proliferazione, sopravvivenza e differenziamento di due inibitori delle DNMTs (DNMTi) non nucleosidici di nuova sintesi, MC 3353 e MC 3343, in comparazione alla DAC, su linee e xenotrapianti di cellule OS ed EWS. Entrambi gli inibitori hanno determinato una significativa inibizione dell'attività DNA metil-transferasica, ma solo il trattamento con MC 3343 ha influenzato l'espressione delle DNMTs. In vitro, MC 3343 ha dimostrato effetti specifici su proliferazione e differenziamento, mentre MC 3353 ha comportato un generale effetto tossico. MC 3343 rallenta il ciclo cellulare con aumento delle cellule nelle fasi G1 o G2/M e induce il differenziamento. In EWS viene indotta l'espressione di marcatori neurali del differenziamento terminale, mentre in OS, si assiste ad un aumento della matrice mineralizzata e dell’espressione di geni dell’osteoblastogenesi matura. Il trattamento in vivo di uno xenotrapianto derivato da paziente OS (PDX) ha confermato l'effetto antiproliferativo di MC 3343 insieme ad una chiara induzione del differenziamento osteoblastico. In conclusione, MC 3343 ha dimostrato specificità ed efficacia nel bloccare la proliferazione e indurre il differenziamento sia nel contesto EWS che OS. Inoltre, la combinazione con doxorubicina e cisplatino è risultata sinergica. Questo composto può quindi rappresentare un potenziale strumento per la terapia differenziativa dei sarcomi ossei.Identification of new therapeutic strategies against bone sarcomas continues to represent a primary goal for patients refractory to common chemotherapy treatments. Both osteosarcoma (OS) and Ewing’s sarcoma (EWS), the two most common primary bone tumors, derive from transformation of mesenchymal stem cells (MSCs) and/or osteoblastic progenitors in any phases of their differentiation. The loss of differentiation is a widespread biological feature of these tumors and has a strong prognostic significance. Thus, reverting epigenetic alterations to restore differentiation may be a therapeutic opportunity. Here we analyze the effects of two novel non-nucleoside DNMT inhibitors (DNMTi), MC 3353 and MC 3343, in comparison with DAC nucleoside inhibitor, on cell proliferation, survival and differentiation, in a representative panel of OS and EWS cell lines and xenografts. Both inhibitors determined a significant inhibition of DNMTs activity, but only MC 3343 treatment affected the expression of DNMTs. In vitro, MC 3343 was found to have specific effects on cell proliferation and differentiation, while MC 3353 induced a general toxic effect. MC 3343 altered cell cycle by inducing blockage of cells in G1 or G2/M phases and was able to significantly induce cell differentiation. In EWS, the treatment induced the expression of terminal neural differentiation markers, while in OS cells, increased both matrix mineralization and expression of genes specifically related to osteoblastogenesis. In vivo treatment of an OS patient derived xenograft (PDX) confirmed the anti-proliferative effect of MC 3343 together with the severe induction of osteoblastic differentiation. In conclusion, MC 3343 non-nucleoside DNMTi, was found to be specific and effective to reduce proliferation and induce differentiation either in EWS or in OS context and its combination with doxorubicin and cisplatin, two of the chemotherapeutic agents of choice in OS treatment, resulted synergic. This compound may thus be considered for differentiative therapy of bone sarcomas

ROCK2 deprivation leads to the inhibition of tumor growth and metastatic potential in osteosarcoma cells through the modulation of YAP activity

The treatment of metastatic osteosarcoma (OS) remains a challenge for oncologists, and novel therapeutic strategies are urgently needed. An understanding of the pathways that regulate OS dissemination is required for the design of novel treatment approaches. We recently identified Rho-associated coiled-coil containing protein kinase 2 (ROCK2) as a crucial driver of OS cell migration. In this study, we explored the impact of ROCK2 disruption on the metastatic capabilities of OS cells and analyzed its functional relationship with Yes-associated protein-1 (YAP), the main transcriptional mediator of mechanotransduction signaling

Novel Targeting of DNA Methyltransferase Activity Inhibits Ewing Sarcoma Cell Proliferation and Enhances Tumor Cell Sensitivity to DNA Damaging Drugs by Activating the DNA Damage Response

DNA methylation is an important component of the epigenetic machinery that regulates the malignancy of Ewing sarcoma (EWS), the second most common primary bone tumor in children and adolescents. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming and the DNMT1 enzyme has been demonstrated to have an important role in both maintaining the epigenome and controlling cell cycle. Here, we showed that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 induces a specific depletion of DNMT1 and affects EWS tumor proliferation through a mechanism that is independent on DNA methylation. Depletion of DNMT1 causes perturbation of the cell cycle, with an accumulation of cells in the G1 phase, and DNA damage, as revealed by the induction of gamma H2AX foci. These effects elicited activation of p53-dependent signaling and apoptosis in p53wt cells, while in p53 mutated cells, persistent micronuclei and increased DNA instability was observed. Treatment with MC3343 potentiates the efficacy of DNA damaging agents such as doxorubicin and PARP-inhibitors (PARPi). This effect correlates with increased DNA damage and synergistic tumor cytotoxicity, supporting the use of the DNMTi MC3343 as an adjuvant agent in treating EWS

Engagement of CD99 activates distinct programs in Ewing sarcoma and macrophages

Ewing sarcoma (EWS) is the second most common pediatric bone tumor. The EWS tumor microenvironment is largely recognized as immune-cold, with macrophages being the most abundant immune cells and their presence associated with worse patient prognosis. Expression of CD99 is a hallmark of EWS cells, and its targeting induces inhibition of EWS tumor growth through a poorly understood mechanism. In this study, we analyzed CD99 expression and functions on macrophages and investigated whether the concomitant targeting of CD99 on both tumor and macrophages could explain the inhibitory effect of this approach against EWS. Targeting CD99 on EWS cells downregulated expression of the "don't eat-me" CD47 molecule but increased levels of the "eat-me" phosphatidyl serine and calreticulin molecules on the outer leaflet of the tumor cell membrane, triggering phagocytosis and digestion of EWS cells by macrophages. In addition, CD99 ligation induced reprogramming of undifferentiated M0 macrophages and M2-like macrophages toward the inflammatory M1-like phenotype. These events resulted in the inhibition of EWS tumor growth. Thus, this study reveals what we believe to be a previously unrecognized function of CD99, which engenders a virtuous circle that delivers intrinsic cell death signals to EWS cells, favors tumor cell phagocytosis by macrophages, and promotes the expression of various molecules and cytokines, which are pro-inflammatory and usually associated with tumor regression. This raises the possibility that CD99 may be involved in boosting the antitumor activity of macrophages

Correction to: ABCA6 affects the malignancy of Ewing sarcoma cells via cholesterol-guided inhibition of the IGF1R/AKT/MDM2 axis

In this article an incorrect affiliation had inadvertently been added. The original article has been updated

Bone sarcoma patient-derived xenografts are faithful and stable preclinical models for molecular and therapeutic investigations

Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient's tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches

A 3D Collagen-Based Bioprinted Model to Study Osteosarcoma Invasiveness and Drug Response

The biological and therapeutic limits of traditional 2D culture models, which only partially mimic the complexity of cancer, have recently emerged. In this study, we used a 3D bioprinting platform to process a collagen-based hydrogel with embedded osteosarcoma (OS) cells. The human OS U-2 OS cell line and its resistant variant (U-2OS/CDDP 1 μg) were considered. The fabrication parameters were optimized to obtain 3D printed constructs with overall morphology and internal microarchitecture that accurately match the theoretical design, in a reproducible and stable process. The biocompatibility of the 3D bioprinting process and the chosen collagen bioink in supporting OS cell viability and metabolism was confirmed through multiple assays at short- (day 3) and long- (day 10) term follow-ups. In addition, we tested how the 3D collagen-based bioink affects the tumor cell invasive capabilities and chemosensitivity to cisplatin (CDDP). Overall, we developed a new 3D culture model of OS cells that is easy to set up, allows reproducible results, and better mirrors malignant features of OS than flat conditions, thus representing a promising tool for drug screening and OS cell biology research

ABCA6 affects the malignancy of Ewing sarcoma cells via cholesterol-guided inhibition of the IGF1R/AKT/MDM2 axis

Purpose The relevance of the subfamily A members of ATP-binding cassette (ABCA) transporters as biomarkers of risk and response is emerging in different tumors, but their mechanisms of action have only been partially defined. In this work, we investigated their role in Ewing sarcoma (EWS), a pediatric cancer with unmet clinical issues. Methods The expression of ABC members was evaluated by RT-qPCR in patients with localized EWS. The correlation with clinical outcome was established in different datasets using univariate and multivariate statistical methods. Functional studies were conducted in cell lines from patient-derived xenografts (PDXs) using gain- or loss-of-function approaches. The impact of intracellular cholesterol levels and cholesterol lowering drugs on malignant parameters was considered. Results We found that ABCA6, which is usually poorly expressed in EWS, when upregulated became a prognostic factor of a favorable outcome in patients. Mechanistically, high expression of ABCA6 impaired cell migration and increased cell chemosensitivity by diminishing the intracellular levels of cholesterol and by constitutive IGF1R/AKT/mTOR expression/activation. Accordingly, while exposure of cells to exogenous cholesterol increased AKT/mTOR activation, the cholesterol lowering drug simvastatin inhibited IGF1R/AKT/mTOR signaling and prevented Ser166 phosphorylation of MDM2. This, in turn, favored p53 activation and enhanced pro-apoptotic effects of doxorubicin. Conclusions Our study reveals that ABCA6 acts as tumor suppressor in EWS cells via cholesterol-mediated inhibition of IGF1R/AKT/MDM2 signaling, which promotes the pro-apoptotic effects of doxorubicin and reduces cell migration. Our findings also support a role of ABCA6 as biomarker of EWS progression and sustains its assessment for a more rational use of statins as adjuvant drugs

Novel Targeting of DNA Methyltransferase Activity Inhibits Ewing Sarcoma Cell Proliferation and Enhances Tumor Cell Sensitivity to DNA Damaging Drugs by Activating the DNA Damage Response

The research leading to these results has received funding from AIRC under IG 2019 - ID. 22805 project - P.I., Katia Scotlandi, and Ministry of University and Research under FISR2019_00374 MeDyCa project - P.I. Antonello Mai. The authors would also thank Cristina Ghinelli for graphic support.Altres ajuts: This work was supported by the Italian Ministry of Health (RF-2016-02361373).DNA methylation is an important component of the epigenetic machinery that regulates the malignancy of Ewing sarcoma (EWS), the second most common primary bone tumor in children and adolescents. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming and the DNMT1 enzyme has been demonstrated to have an important role in both maintaining the epigenome and controlling cell cycle. Here, we showed that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 induces a specific depletion of DNMT1 and affects EWS tumor proliferation through a mechanism that is independent on DNA methylation. Depletion of DNMT1 causes perturbation of the cell cycle, with an accumulation of cells in the G1 phase, and DNA damage, as revealed by the induction of γH2AX foci. These effects elicited activation of p53-dependent signaling and apoptosis in p53wt cells, while in p53 mutated cells, persistent micronuclei and increased DNA instability was observed. Treatment with MC3343 potentiates the efficacy of DNA damaging agents such as doxorubicin and PARP-inhibitors (PARPi). This effect correlates with increased DNA damage and synergistic tumor cytotoxicity, supporting the use of the DNMTi MC3343 as an adjuvant agent in treating EWS