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The RAS/MEK/ERK genetic axis is commonly altered in rhabdomyosarcoma (RMS), indicating high activity of downstream effector ERK1/2 kinase. Previously, we have demonstrated that inhibition of the RAS/MEK/ERK signaling pathway in RMS is insufficient to induce cell death due to residual pro-survival MCL-1 activity. Here, we show that the combination of ERK1/2 inhibitor Ulixertinib and MCL-1 inhibitor S63845 is highly synergistic and induces apoptotic cell death in RMS in vitro and in vivo. Importantly, Ulixertinib/S63845 co-treatment suppresses long-term survival of RMS cells, induces rapid caspase activation and caspase-dependent apoptosis. Mechanistically, Ulixertinib-mediated upregulation of BIM and BMF in combination with MCL-1 inhibition by S63845 shifts the balance of BCL-2 proteins towards a pro-apoptotic state resulting in apoptosis induction. A genetic silencing approach reveals that BIM, BMF, BAK and BAX are all required for Ulixertinib/S63845-induced apoptosis. Overexpression of BCL-2 rescues cell death triggered by Ulixertinib/S63845 co-treatment, confirming that combined inhibition of ERK1/2 and MCL-1 effectively induces cell death of RMS cells via the intrinsic mitochondrial apoptotic pathway. Thus, this study is the first to demonstrate the cytotoxic potency of co-inhibition of ERK1/2 and MCL-1 for RMS treatment., (Copyright © 2021. Published by Elsevier Inc.)

BH3 mimetics are promising novel anticancer therapeutics. By selectively inhibiting BCL-2, BCL-x L , or MCL-1 (i.e. ABT-199, A-1331852, S63845) they shift the balance of pro- and anti-apoptotic proteins in favor of apoptosis. As Bromodomain and Extra Terminal (BET) protein inhibitors promote pro-apoptotic rebalancing, we evaluated the potential of the BET inhibitor JQ1 in combination with ABT-199, A-1331852 or S63845 in rhabdomyosarcoma (RMS) cells. The strongest synergistic interaction was identified for JQ1/A-1331852 and JQ1/S63845 co-treatment, which reduced cell viability and long-term clonogenic survival. Mechanistic studies revealed that JQ1 upregulated BIM and NOXA accompanied by downregulation of BCL-x L , promoting pro-apoptotic rebalancing of BCL-2 proteins. JQ1/A-1331852 and JQ1/S63845 co-treatment enhanced this pro-apoptotic rebalancing and triggered BAK- and BAX-dependent apoptosis since a) genetic silencing of BIM, BAK or BAX, b) inhibition of caspase activity with zVAD.fmk and c) overexpression of BCL-2 all rescued JQ1/A-1331852- and JQ1/S63845-induced cell death. Interestingly, NOXA played a different role in both treatments, as genetic silencing of NOXA significantly rescued from JQ1/A-1331852-mediated apoptosis but not from JQ1/S63845-mediated apoptosis. In summary, JQ1/A-1331852 and JQ1/S63845 co-treatment represent new promising therapeutic strategies to synergistically trigger mitochondrial apoptosis in RMS., Competing Interests: Declaration of Competing Interest None to declare., (Copyright © 2021. Published by Elsevier Inc.)

The induction of apoptosis is a direct way to eliminate tumor cells and improve cancer therapy. Apoptosis is tightly controlled by the balance of pro- and antiapoptotic Bcl-2 proteins. BH3 mimetics neutralize the antiapoptotic function of Bcl-2 proteins and are highly promising compounds inducing apoptosis in several cancer entities including pediatric malignancies. However, the clinical application of BH3 mimetics in solid tumors is impeded by the frequent resistance to single BH3 mimetics and the anticipated toxicity of high concentrations or combination treatments. One potential avenue to increase the potency of BH3 mimetics is the development of immune cell-based therapies to counteract the intrinsic apoptosis resistance of tumor cells and sensitize them to immune attack. Here, we describe spheroid cultures of pediatric cancer cells that can serve as models for drug testing. In these 3D models, we were able to demonstrate that activated allogeneic Natural Killer (NK) cells migrated into tumor spheroids and displayed cytotoxicity against a wide range of pediatric cancer spheroids, highlighting their potential as anti-tumor effector cells. Next, we investigated whether treatment of tumor spheroids with subtoxic concentrations of BH3 mimetics can increase the cytotoxicity of NK cells. Notably, the cytotoxic effects of NK cells were enhanced by the addition of BH3 mimetics. Treatment with either the Bcl-X L inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-X L or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy., (© 2022. The Author(s).)

Targeting the epigenome of cancer cells with the combination of Bromodomain and Extra Terminal (BET) protein inhibitors and histone deacetylase (HDAC) inhibitors has shown synergistic antitumor effects in several cancer types. In this study, we investigate the antitumor potential of the novel dual BET/HDAC inhibitor TW09 in rhabdomyosarcoma (RMS) cells. TW09 reduces cell viability, suppresses long-term clonogenic survival and induces cell death in RMS cells in a dose-dependent manner. Compared to BET/HDAC co-inhibition using JQ1 and MS-275, TW09 induces similar cell death at equimolar concentrations and regulates BET and HDAC target proteins (e.g. c-MYC, H3 acetylation). Mechanistic studies revealed that TW09 upregulates BIM, NOXA, PUMA and BMF, while downregulating BCL-X L , leading to proapoptotic rebalancing of BCL-2 proteins. This results in BAK and BAX activation and caspase-dependent apoptosis, since individual genetic silencing of BIM, NOXA, PUMA, BMF, BAK or BAX, overexpression of BCL-2 or the caspase inhibition with zVAD.fmk all rescue JQ1/BYL719-induced cell death. In conclusion, TW09 shows potent antitumor activity in RMS cells in vitro by inducing mitochondrial apoptosis and may represent a promising new therapeutic option for the treatment of RMS., Competing Interests: Declaration of competing interest None to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Remodeling transcription by targeting bromodomain and extraterminal (BET) proteins has emerged as promising anticancer strategy. Here, we identify a novel synergistic interaction of the BET inhibitor JQ1 with the PI3Kα-specific inhibitor BYL719 to trigger mitochondrial apoptosis and to suppress tumor growth in models of rhabdomyosarcoma (RMS). RNA-Seq revealed that JQ1/BYL719 co-treatment shifts the overall balance of BCL-2 family gene expression towards apoptosis and upregulates expression of BMF, BCL2L11 (BIM), and PMAIP1 (NOXA) while downregulating BCL2L1 (BCL-x L ). These changes were confirmed by qRT-PCR and western blot analysis. Ingenuity pathway analysis (IPA) of RNA-Seq data followed by validation qRT-PCR and western blot identified MYC and FOXO3a as potential transcription factors (TFs) upstream of the observed gene expression pattern. Immunoprecipitation (IP) studies showed that JQ1/BYL719-stimulated increase in BIM expression enhances the neutralization of antiapoptotic BCL-2, BCL-x L , and MCL-1. This promotes the activation of BAK and BAX and caspase-dependent apoptosis, as (1) individual silencing of BMF, BIM, NOXA, BAK, or BAX, (2) overexpression of BCL-2 or MCL-1 or (3) the caspase inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (zVAD.fmk) all rescue JQ1/BYL719-induced cell death. In conclusion, co-inhibition of BET proteins and PI3Kα cooperatively induces mitochondrial apoptosis by proapoptotic re-balancing of BCL-2 family proteins. This discovery opens exciting perspectives for therapeutic exploitation of BET inhibitors in RMS.

In the present study, we show that concomitant inhibition of Hedgehog (HH) signaling by the glioma-associated oncogene homolog1 (GLI1)-targeting agent GANT61 and the antiapoptotic BCL-2 protein family member MCL-1 by A-1210477 synergistically induces cell death in HH-driven cancers, i.e. rhabdomyosarcoma (RMS) and medulloblastoma (MB) cells. Combined genetic and pharmacological inhibition emphasized that co-treatment of GANT61 and A-1210477 indeed relies on inhibition of GLI1 (by GANT61) and MCL-1 (by A-1210477). Mechanistic studies revealed that A-1210477 triggers the release of BIM from MCL-1 and its shuttling to BCL-x L and BCL-2. Indeed, BIM proved to be required for GANT61/A-1210477-induced cell death, as genetic silencing of BIM using siRNA significantly rescues cell death upon GANT61/A-1210477 co-treatment. Similarly, genetic silencing of NOXA results in a significant reduction of GANT61/A-1210477-mediated cell death. Also, overexpression of MCL-1 or BCL-2 significantly protects RMS cells from GANT61/A-1210477-triggered cell death. Addition of the pan-caspase inhibitor zVAD.fmk significantly decreases GANT61/A-1210477-stimulated cell demise, indicating apoptotic cell death. In conclusion, GANT61 and A-1210477 synergize to engage mitochondrial apoptosis. These findings provide the rationale for further evaluation of dual inhibition of HH signaling and MCL-1 in HH-driven cancers., (Copyright © 2019. Published by Elsevier B.V.)

In the present study, we show that pharmacological repression by the Hedgehog (Hh) pathway inhibitor (HPI) GANT61 induces expression of the proapoptotic protein Noxa in TP53-mutated embryonal pediatric tumor cells driven by Hh signaling (i.e. rhabdomyosarcoma (RMS) and medulloblastoma (MB)). Similarly, genetic silencing of Gli1 by siRNA causes increased Noxa mRNA and protein levels, while overexpression of Gli1 results in decreased Noxa expression. Furthermore, TAp73 mRNA and protein levels are increased upon Gli1 knockdown, while Gli1 overexpression reduces TAp73 mRNA and protein levels. However, knockdown of TAp73 fails to block Noxa induction in GANT61-treated cells, suggesting that Noxa is not primarily regulated by TAp73. Interestingly, mRNA levels of the transcription factor EGR1 correlate with those of Noxa and TAp73. Silencing of EGR1 results in decreased Noxa and TAp73 mRNA levels, indicating that EGR1 is involved in regulating transcriptional activity of Noxa and TAp73. These findings suggest that Gli1 represses Noxa and TAp73, possibly via EGR1. These findings could be exploited for the treatment of Hh-driven tumors, e.g. for their sensitization to chemotherapeutic agents., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Histone acetylation marks exert essential functions in regulating gene expression. These marks are written by histone acetyltransferases (HATs), removed by histone deacetylases (HDACs) and read by e.g. BET proteins. While BET inhibitors are promising new anticancer drugs, little is yet known about their antitumor activity in rhabdomyosarcoma (RMS). We therefore investigated the efficacy of the prototypic BET inhibitor JQ1 alone or in combination with other epigenetic modifiers, namely HDAC inhibitors (HDACIs). Here, we discover a synergistic interaction of the panBET inhibitor JQ1 together with various HDACIs, i.e. Quisinostat (JNJ-26481585), Vorinostat (SAHA), Entinostat (MS-275) and Panobinostat (LBH589), inducing apoptosis in RMS cells, whereas JQ1 as single agent exhibits little cytotoxicity. Calculation of combination index (CI) confirmed the synergism of this combination. Importantly, JQ1 and JNJ-26481585 act in concert to suppress colony formation and to trigger apoptosis in an in vivo model. Mechanistic studies revealed that combination of JQ1 and JNJ-26481585 cooperatively upregulates BIM and BMF, while downregulating BCL-x L . This shifted ratio of pro- and antiapoptotic BCL-2 proteins engages activation of BAX and BAK and increases caspases-3 and -7 activity. Individual silencing of BIM or NOXA, overexpression of BCL-2 or MCL-1 as well as addition of the caspase inhibitor zVAD.fmk significantly rescue JQ1/JNJ-26481585-induced apoptosis. Thus, co-targeting of histone acetylation by concomitant inhibition of HDAC and BET proteins synergistically induces mitochondrial apoptosis by shifting the ratio of pro- and antiapoptotic BCL-2 proteins towards apoptosis. These findings indicate that combinatorial use of BET and HDACIs may represent a promising new strategy for the treatment of RMS., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Rhabdomyosarcoma (RMS), the most common cancer of connective tissues in pediatrics, is often resistant to conventional therapies. One underlying mechanism of this resistance is the overexpression of Inhibitor of Apoptosis (IAP) proteins, leading to a dysfunctional cell death program within tumor cells. Smac mimetics (SM) are small molecules that can reactivate the cell death program by antagonizing IAP proteins and thereby compensating their overexpression. Here, we report that SM sensitize two RMS cell lines (RD and RH30) toward natural killer (NK) cell-mediated killing on the one hand, and increase the cytotoxic potential of NK cells on the other. The SM-induced sensitization of RH30 cells toward NK cell-mediated killing is significantly reduced through blocking tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on NK cells prior to coculture. In addition, the presence of zVAD.fmk, a pancaspase inhibitor, rescues tumor cells from the increase in killing, indicating an apoptosis-dependent cell death. On the NK cell side, the presence of SM in addition to IL-2 during the ex vivo expansion leads to an increase in their cytotoxic activity against RH30 cells. This effect is mainly TNFα-dependent and partially mediated by NK cell activation, which is associated with transcriptional upregulation of NF-κB target genes such as IκBα and RelB. Taken together, our findings implicate that SM represent a novel double-hit strategy, sensitizing tumor and activating NK cells with one single drug.

The prognosis of metastatic or relapsed rhabdomyosarcoma (RMS) is poor, highlighting the need of new treatment options. In the present study, we evaluated the in vitro efficacy of arsenic trioxide (ATO) in RMS, a FDA-approved drug used in pediatric leukemia. Here, we report that ATO exerts antitumor activity against RMS cells both as single agent and in combination with microtubule-targeting drugs. Monotherapy with ATO reduces cell viability, triggers apoptosis and suppresses clonogenic survival of RMS cells, at least in part, by transcriptional induction of the proapoptotic BH3-only protein Noxa. siRNA-mediated knockdown of Noxa significantly rescues ATO-mediated cell death, demonstrating that Noxa is required for cell death. Also, ATO suppresses endogenous Hedgehog (Hh) signaling, as it significantly reduces Gli1 transcriptional activity and expression levels of several Hh target genes. Furthermore, we identify synergistic induction of apoptosis by ATO together with several antimicrotubule agents including vincristine (VCR), vinblastine and eribulin. The addition of the broad-range caspase inhibitor zVAD.fmk or overexpression of the antiapoptotic protein Bcl-2 significantly reduce ATO/VCR-induced cell death, indicating that the ATO/VCR combination triggers caspase-dependent apoptosis via the mitochondrial pathway. In summary, ATO exerts antitumor activity against RMS, especially in combination with antimicrotubule drugs. These findings have important implications for the development of novel therapeutic strategies for RMS., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

A range of studies has addressed possible environmental impacts of nanosilver, but most focused on acute effects in few species. Moreover, it remains unclear if toxic effects are particle-specific or mediated by released silver ions. We investigated chronic effects of nanosilver and soluble silver (AgNO3) on the freshwater bivalve Sphaerium corneum. Animals were exposed to nanosilver (0-500 μg Ag L(-1)) and AgNO3 (0-318 μg Ag L(-1)) over 28 days, and effects on reproduction and behavioral changes were assessed. To explore toxic mechanisms, we evaluated the effects on intracellular levels of reactive oxygen species (ROS) and the activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase). We further explored the activity of the sodium-potassium adenosine triphosphatase (Na(+)/K(+)-ATPase). Chronic exposure to nanosilver and AgNO3 resulted in negative effects on reproduction at concentrations of 5 and 3.18 µg Ag L(-1) (LOEC), respectively. ROS levels significantly increased after exposure to nanosilver at 10 µg Ag L(-1) and AgNO3 at 63.5 µg Ag L(-1). Both forms of silver altered the activities of antioxidant enzymes. Nanosilver (500 μg Ag L(-1)) and AgNO3 (318 μg Ag L(-1)) inhibited Na(+)/K(+)-ATPase activity by 82.6 and 78.9%, respectively. Nanoparticulate and soluble silver produced similar effects in S. corneum suggesting that toxicity of nanosilver is mainly mediated by dissolution of nanoparticles in the test media or after uptake by the test organisms.

The antibacterial properties of nanosilver have led to a versatile application spectrum including medical purposes and personal care products. However, the increasing use of nanosilver has raised concerns about its environmental impacts. Long-term exposure studies with aquatic invertebrates are essential to assess possible adverse effects on aquatic ecosystems. In the present study, acute (48 h), chronic (21 d) and long-term effects of nanosilver (primary size 15 nm) on five successive generations of three Daphnia species (D. magna, D. pulex, and D. galeata) were investigated. Acute EC50 values of nanosilver were 121 µg Ag L(-1) for D. magna being the least sensitive species and 8.95 and 13.9 µg Ag L(-1) for D. pulex and D. galeata, respectively. Chronic exposure provided EC10 values of 0.92 µg Ag L(-1) for D. magna showing the most sensitive chronic reaction and 2.25 and 3.45 µg Ag L(-1) for D. pulex and D. galeata, respectively. Comparative exposure to AgNO3 revealed a generally higher toxicity of the soluble form of silver. The multi-generation experiments resulted in effects on the population level for all tested species. Exposure of D. magna indicated an increased toxicity of nanosilver in the fifth generation of animals exposed to 10 µg Ag L(-1). Neonates from pre-exposed parental daphnids did not completely recover when transferred into clean water. Exposure of D. pulex and D. galeata revealed not only increasing toxicity in some generations, but also greater tolerance to nanosilver. This study contributes to the assessment of the risk potential of nanosilver on aquatic ecosystems. It shows that effects of nanosilver vary within one genus and change with exposure duration. Therefore, long-term studies considering different aquatic species are needed to better understand the possible effects of nanosilver on aquatic ecosystems.