Mcl‐1 targeting strategies unlock the proapoptotic potential of TRAIL in melanoma cells

TNF‐related apoptosis‐inducing ligand (TRAIL) induces apoptosis selectively in cancer cells. For melanoma, the targeting of TRAIL signaling appears highly attractive, due to pronounced TRAIL receptor expression in tumor tissue. However, mechanisms of TRAIL resistance observed in melanoma cells may limit its clinical use. The Bcl‐2 family members are critical regulators of cell‐intrinsic apoptotic pathways. Thus, the antiapoptotic Bcl‐2 protein myeloid cell leukemia 1 (Mcl‐1) is overexpressed in many tumor types and was linked to chemotherapy resistance in melanoma. In this study, we evaluated the involvement of antiapoptotic Bcl‐2 proteins (Bcl‐2, Bcl‐xL, Bcl‐w, Mcl‐1, Bcl‐A1, and Bcl‐B) in TRAIL resistance. They were targeted by small interfering RNA‐mediated silencing in TRAIL‐sensitive (A‐375, Mel‐HO) and in TRAIL‐resistant melanoma cell lines (Mel‐2a, MeWo). This highlighted Mcl‐1 as the most efficient target to overcome TRAIL resistance. In this context, we investigated the effects of Mcl‐1‐targeting microRNAs as well as the Mcl‐1‐selective inhibitor S63845. Both miR‐193b and S63845 resulted in significant enhancement of TRAIL‐induced apoptosis, associated with decreased cell viability. Apoptosis induction was mediated by caspase‐3 processing as well as by Bax and Bak activation, indicating the critical involvement of intrinsic apoptosis pathways. These data may indicate a high relevance of Mcl‐1 targeting also in melanoma therapy. Furthermore, the data may suggest to consider the use of the tumor suppressor miR‐193b as a strategy for countering TRAIL resistance in melanoma

Inhibition of SARS-CoV-2 Replication by a Small Interfering RNA Targeting the Leader Sequence

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected almost 200 million people worldwide and led to approximately 4 million deaths as of August 2021. Despite successful vaccine development, treatment options are limited. A promising strategy to specifically target viral infections is to suppress viral replication through RNA interference (RNAi). Hence, we designed eight small interfering RNAs (siRNAs) targeting the highly conserved 5′-untranslated region (5′-UTR) of SARS-CoV-2. The most promising candidate identified in initial reporter assays, termed siCoV6, targets the leader sequence of the virus, which is present in the genomic as well as in all subgenomic RNAs. In assays with infectious SARS-CoV-2, it reduced replication by two orders of magnitude and prevented the development of a cytopathic effect. Moreover, it retained its activity against the SARS-CoV-2 alpha variant and has perfect homology against all sequences of the delta variant that were analyzed by bioinformatic means. Interestingly, the siRNA was even highly active in virus replication assays with the SARS-CoV-1 family member. This work thus identified a very potent siRNA with a broad activity against various SARS-CoV viruses that represents a promising candidate for the development of new treatment options

Silencing of Mcl-1 overcomes resistance of melanoma cells against TRAIL-armed oncolytic adenovirus by enhancement of apoptosis

Arming of oncolytic viruses with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown as a viable approach to increase the antitumor efficacy in melanoma. However, melanoma cells may be partially or completely resistant to TRAIL or develop TRAIL resistance, thus counteracting the antitumor efficiency of TRAIL-armed oncolytic viruses. Recently, we found that TRAIL resistance in melanoma cells can be overcome by inhibition of antiapoptotic Bcl-2 protein myeloid cell leukemia 1 (Mcl-1). Here, we investigated whether the cytotoxicity of AdV-TRAIL, an oncolytic adenovirus, which expresses TRAIL after induction by doxycycline (Dox), can be improved in melanoma cells by silencing of Mcl-1. Two melanoma cell lines, the TRAIL-resistant MeWo and the TRAIL-sensitive Mel-HO were investigated. Treatment of both cell lines with AdV-TRAIL resulted in a decrease of cell viability, which was caused by an increase of apoptosis and necrosis. The proapoptotic effects were dependent on induction of TRAIL by Dox and were more pronounced in Mel-HO than in MeWo cells. SiRNA-mediated silencing of Mcl-1 resulted in a further significant decrease of cell viability and a further increase of apoptosis and necrosis in AdV-TRAIL-infected MeWo and Mel-HO cells. However, while in absolute terms, the effects were more pronounced in Mel-HO cells, in relative terms, they were stronger in MeWo cells. These results show that silencing of Mcl-1 represents a suitable approach to increase the cytotoxicity of a TRAIL-armed oncolytic adenovirus in melanoma cells.TU Berlin, Open-Access-Mittel – 202

Improvement of the cytotoxicity of a TRAIL-expressing oncolytic adenovirus in melanoma cells by knockdown of the antiapoptotic protein Mcl-1

Die Inzidenz des malignen Melanoms hat in den letzten Jahrzehnten zugenommen. Weltweit wird jedes Jahr bei ungefähr 300.000 Patienten ein Melanom diagnostiziert. Das Melanom gehört damit zu den zehn häufigsten Tumorarten in Nordamerika, Westeuropa und Ozeanien. In den letzten Jahren hat die Zulassung von selektiven Inhibitoren für MAP-Kinasen BRAF und MEK sowie von Immuncheckpoint-Inhibitoren, wie beispielsweise Antikörper gegen PD-1 und CTLA-4, die Melanomtherapie signifikant verbessert. Dennoch sind die oft frühzeitige Metastasierung und die hohe Therapieresistenz von Melanomen dafür verantwortlich, dass nach 5 Jahren nur 25 % der Patienten überleben, bei denen Metastasen aufgetreten sind. Deshalb besteht weiterhin der dringende Bedarf, neue Therapien zu entwickeln. Ein vielversprechendes neues Behandlungskonzept ist der Einsatz von onkolytischen Viren, die selektiv in Tumorzellen replizieren und diese dadurch zerstören. Bisher wurden die zwei onkolytischen Viren Rigvir und T-VEC für die Behandlung von Melanomen zugelassen. Um die Effizienz von onkolytischen Viren zu verbessern, werden diese häufig mit Transgenen ausgestattet, die die antitumorale Aktivität der onkolytischen Viren weiter steigert. Da gezeigt werden konnte, dass eine Apoptoseresistenz häufig ursächlich für die Therapieresistenz von Melanomzellen ist, wurde vor Kurzem das onkolytische Adenovirus Ad-TRAIL entwickelt. Ad-TRAIL ist ein selektiv in Melanomzellen replizierendes onkolytisches Adenovirus, das mit TRAIL unter Kontrolle eines Dox-abhängigen Promotors ausgestattet ist. TRAIL ist ein Mitglied der TNF-Superfamilie und induziert Apoptose in Tumorzellen, während normale Zellen geschützt sind. In in vitro und in vivo Studien mit Ad-TRAIL zeigte sich, dass die Kombination von viraler Onkolyse und selektiver Apoptoseinduktion zu einer signifikant verbesserten antitumoralen Aktivität des Virus in Melanomzellen beitrug. Darüber hinaus wies Ad-TRAIL ein deutlich erhöhtes Sicherheitsprofil auf. Trotz der vielversprechenden Ergebnisse war der Einsatz von Ad-TRAIL jedoch begrenzt, da einige Melanomzellen intrinsisch resistent gegenüber TRAIL sind oder nach längerer Exposition eine Resistenz gegen TRAIL entwickelten. Zahlreiche Studien deuten dabei darauf hin, dass antiapoptotische Bcl-2-Proteine, vor allem Mcl-1, wesentlich für das Überleben von Melanomzellen sind und eine Resistenz gegenüber einer TRAIL-induzierten Apoptose vermitteln. In diesem Zusammenhang verfolgte die vorliegende Arbeit zwei Ziele. Erstens sollte aufgeklärt werden, ob Melanomzellen durch eine Herunterregulation von Mcl-1 gegenüber Ad-TRAIL sensitiviert werden können. Zweitens sollten verschiedene RNA-Interferenz-vermittelnde Sequenzen für den Knockdown von Mcl-1 verglichen werden, um zukünftig die effizienteste Sequenz in das Genom von Ad-TRAIL inserieren zu können und somit die Apoptoseinduktion durch TRAIL sowie die Herunterregulation von Mcl-1 in einem Virus zu vereinen. Die Verbesserung der Effizienz des onkolytischen Adenovirus Ad-TRAIL in resistenten Melanomzellen war dabei das übergeordnete Ziel dieser Arbeit. Zunächst konnte in diesem Zusammenhang die deutlich unterschiedliche Sensitivität von vier Melanomzelllinien gegenüber löslichem TRAIL und Ad-TRAIL bestätigt werden (Mel-HO > SK-Mel-19 > Mel-2a > MeWo). Anschließend wurde untersucht, ob ein Knockdown von Mcl-1 Melanomzellen gegenüber Ad-TRAIL sensitivieren kann. Um dabei den Einfluss der unterschiedlichen Sensitivitäten der Melanomzelllinien vergleichen zu können, wurden für diese Experimente zum einen die TRAIL-resistenten MeWo- und zum anderen die TRAIL-sensitiven Mel-HO-Zellen eingesetzt. Es konnte gezeigt werden, dass die allgemeine zytolytische Aktivität, die Apoptoseinduktion und die Nekroseinduktion von Ad-TRAIL signifikant durch eine Herunterregulation von Mcl-1 gesteigert werden konnte, insbesondere wenn die TRAIL-Expression durch Dox induziert wurde. Weiterhin konnte gezeigt werden, dass die erhöhte Apoptoseinduktion durch TRAIL in Kombination mit der Herunterregulierung von Mcl-1 mit einer stärkeren Aktivierung der Effektor-Caspase-3 einhergeht. Der positive Effekt des Knockdowns von Mcl-1 wurde in der TRAIL-resistenten Zelllinie MeWo, aber auch in der TRAIL-sensitiven Zelllinie Mel-HO beobachtet. Dies zeigt, dass die Vorteile der Herunterregulierung von Mcl-1 nicht auf Melanomzellen mit TRAIL-Resistenz beschränkt sind. Relativ gesehen war der positive Effekt der Herunterregulation von Mcl-1 jedoch in MeWo-Zellen stärker als in Mel-HO-Zellen. Beim Vergleich der verschiedenen RNA-Interferenz-vermittelnden Sequenzen für den Knockdown von Mcl-1 stellte sich die Dreifachkopie einer amiRNA gegen Mcl-1 (3xamiRMcl-1) im Vergleich zu den anderen untersuchten miRNAs und shRNAs als am effizientesten und sichersten für den Knockdown von Mcl-1 heraus. Zusammenfassend legen diese Daten daher eine Herunterregulierung von Mcl-1 als Strategie zur Verbesserung von Ad-TRAIL nahe. In Zukunft könnte in das Genom von Ad-TRAIL die 3xamiRMcl-1-Sequenz inseriert werden, um die induzierbare Expression von TRAIL und die Herunterregulation von Mcl-1 durch RNA-Interferenz in einem Virus zu vereinen.The incidence of malignant melanoma has been increasing over the past decades. Globally each year approximately 300,000 new patients are diagnosed with cutaneous melanoma, which thus belongs to the ten most common cancers in North America, Western Europe and Oceania. Only recently, selective inhibitors for the MAP kinases BRAF and MEK as well as immune checkpoint inhibitors, like PD-1 or CTLA-4 antibodies, have significantly improved melanoma therapy. However, since melanoma is often characterized by early dissemination and severe therapy resistance the current survival rate of patients with metastases still does not exceed 25 % after 5 years. Hence, new strategies to further improve melanoma therapy are required. A promising new strategy is the use of oncolytic adenoviruses, which selectively replicate in and destroy tumor cells. So far, the two oncolytic viruses Rigvir and T-VEC have been approved for the treatment of melanoma. Arming of oncolytic viruses with transgenes, that support the antitumoral activity, has been shown to be a valuable approach to increase the oncolytic efficacy of these viruses. As apoptosis deficiency plays a major role in therapy resistance of melanoma, previously the oncolytic adenovirus Ad-TRAIL was developed. Ad-TRAIL selectively replicates in melanoma cells and is armed with TRAIL under control of a Dox-dependent promoter. TRAIL is a member of the TNF superfamily, that induces apoptosis in tumor cells, while normal cells are largely protected. In vitro and in vivo studies with Ad-TRAIL demonstrated that the combination of viral oncolysis and selective induction of apoptosis contributed to a significantly improved antitumor activity of the virus in melanoma cells. Ad-TRAIL also exhibited a distinctly higher security profile. Despite the promising results the overall efficacy of Ad-TRAIL remained limited, as some melanoma cells were intrinsically resistant to TRAIL or developed resistance to TRAIL after prolonged exposure. Numerous studies indicate that antiapoptotic Bcl-2 proteins - in particular Mcl-1 - are essential for the survival of melanoma cells and mediate resistance to TRAIL-induced apoptosis. Therefore, the present study pursued two aims. First, to investigate whether melanoma cells can be sensitized to Ad-TRAIL by silencing of Mcl-1. Secondly, to compare different RNA interference-mediating sequences for the knockdown of Mcl-1 in order to be able to insert the most efficient sequence into the genome of Ad-TRAIL in the future to thus unite the apoptosis-inducing TRAIL and the silencing of Mcl-1 in one virus. Taken together, the superior aim of this study was to improve the efficiency of the oncolytic adenovirus Ad-TRAIL in resistant melanoma cells. In the present study the different sensitivities of four melanoma cell lines towards soluble TRAIL and Ad-TRAIL could be confirmed (Mel-HO > SK-Mel-19 > Mel-2a > MeWo). Therefore, it was subsequently examined whether a knockdown of Mcl-1 sensitized melanoma cells to Ad-TRAIL. In order to be able to compare the influence of different sensitivities of melanoma cells, the TRAIL-resistant MeWo and the TRAIL-sensitive Mel-HO cells were used for these experiments. It was demonstrated, that the general cytolytic activity, the induction of apoptosis and the induction of necrosis by Ad-TRAIL was significantly increased by the silencing of Mcl-1, in particular when TRAIL expression was induced by Dox. Furthermore, it was shown that the significantly enhanced induction of apoptosis by Ad-TRAIL in combination with the knockdown of Mcl-1 was accompanied by a stronger activation of the main effector caspase-3. The positive effect of Mcl-1 silencing was seen in TRAIL-resistant MeWo, but also in TRAIL-sensitive Mel-HO cells, demonstrating that the benefits of downregulating Mcl-1 were not limited to melanoma cells with TRAIL resistance. However, in relative terms the positive effect of Mcl-1 silencing was stronger in MeWo than in Mel-HO cells. In regard to the evaluation of the different RNA interference-mediating sequences, 3xamiRMcl-1 was found to be the most efficient and safest sequence for the knockdown of Mcl-1 compared to the other miRNAs and shRNAs examined. Taken together, these findings suggest Mcl-1 silencing as a new strategy to further enhance the oncolytic activity of Ad-TRAIL in melanoma. As a future perspective, the 3xamiRMcl-1-sequence could be inserted into the genome of Ad-TRAIL in order to combine the inducible expression of TRAIL and the silencing of Mcl-1 by RNA interference in one virus

Inhibition of SARS-CoV-2 Replication by a Small Interfering RNA Targeting the Leader Sequence

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected almost 200 million people worldwide and led to approximately 4 million deaths as of August 2021. Despite successful vaccine development, treatment options are limited. A promising strategy to specifically target viral infections is to suppress viral replication through RNA interference (RNAi). Hence, we designed eight small interfering RNAs (siRNAs) targeting the highly conserved 5′-untranslated region (5′-UTR) of SARS-CoV-2. The most promising candidate identified in initial reporter assays, termed siCoV6, targets the leader sequence of the virus, which is present in the genomic as well as in all subgenomic RNAs. In assays with infectious SARS-CoV-2, it reduced replication by two orders of magnitude and prevented the development of a cytopathic effect. Moreover, it retained its activity against the SARS-CoV-2 alpha variant and has perfect homology against all sequences of the delta variant that were analyzed by bioinformatic means. Interestingly, the siRNA was even highly active in virus replication assays with the SARS-CoV-1 family member. This work thus identified a very potent siRNA with a broad activity against various SARS-CoV viruses that represents a promising candidate for the development of new treatment options