Molecular mechanisms of Thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene

Molecular mechanisms of Thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular mechanisms of Thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene SARA POZZI, LAURA MONTEONOFRIO, ROBERTO QUADRI, ILARIA VIRDIA, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4162662/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Sep, 2024 Read the published version in Journal of Molecular Medicine → Version 1 posted 5 You are reading this latest preprint version Abstract COVID-19 pandemics is caused by the SARS-CoV-2 virus, whose internalization and infection are mediated by the Angiotensin Converting Enzyme 2 (ACE2). The identification of novel approaches to tackle this step is instrumental for the development of therapies for the management of COVID-19 and other diseases with a similar mechanism of infection. Thalidomide, a drug sadly known for its teratogenic effects, has potent immunomodulatory and anti-inflammatory properties. Treatment with this drug has been shown to improve the immune functions of COVID-19 patients and proposed for the management of COVID-19 in clinical practice through drug repositioning. Here, we investigated the molecular details linking Thalidomide to ACE2 and COVID-19, showing that in conditions mimicking SARS-CoV-2 associated cytokine storm, the transcription factor p63 and ACE2 are stabilized and IL-8 production is increased. In such conditions, we found p63 to bind to and regulate the expression of the ACE2 gene. We previously showed that p63 is degraded upon Thalidomide treatment, and now found that treatment with this drug—or with its analogue Lenalidomide—downregulates ACE2 through p63 degradation. Finally, we found that Thalidomide treatment reduce in vitro infection by pseudo-SARS-CoV-2, a baculovirus pseudotyped with the SARS-CoV-2 spike protein. Overall, we propose the dual effect of Thalidomide in reducing SARS-CoV-2 viral re-entry and inflammation through p63 degradation to weaken SARS-CoV-2 entry into host cells and mitigate lung inflammation, making it a valuable option in clinical management of COVID-19. Thalidomide SARS-CoV-2 ACE2 ΔNp63α COVID-19 Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction SARS-CoV-2 infection, mediated by the binding of the viral Spike (S) protein to the ACE2 receptor in the host cells (1–3) causes the COVID-19 disease. One of the associated complications is a cytokine storm which involves an uncontrolled release of cytokines and chemokines, leading to a systemic inflammatory response linked to lung failure and acute respiratory distress syndrome (ARDS). Given the time and cost involved in developing new therapies, repurposing existing drugs with known profiles has become a cost-effective strategy (4). Therefore, reassessing the efficacy of licensed and experimental drugs has been a go-on choice by the World Health Organization (WHO) and other health agencies to treat emerging health problems. With the initial lack of vaccines and effective agents against SARS-CoV-2, as well as public health emergencies, WHO had identified promising reposition therapies, such as the combination of two anti-HIV drugs ( i.e., lopinavir and ritonavir) or the experimental antiviral compound, remdesivir (5). Furthermore, Thalidomide (Thal), a small molecule drug with many years of history known to cause misery, became a game changer for its multifaceted pharmacological effects such as immunomodulation, anti-inflammation, anti-angiogenesis, and anti-viral effects (6, 7). Several Thal derivatives have been designed, known as Immuno Modulatory drugs (IMids), among which the most used are Lenalidomide (Len) and Pomalidomide, that are approved by FDA for the treatment of several severe diseases like Multiple Myeloma, myelodysplastic syndrome (5q-), mantle cell lymphoma, and follicular lymphoma (8). IMids retain the anti-angiogenic and anti-inflammatory properties of Thal but are devoid of Thal teratogenic activity (6, 8). Extensive information available on Thal’s mechanisms of action, its efficacy and safety in the haemophagocytic syndrome-induced cytokine storm and Idiopathic Pulmonary Fibrosis (IPF) and in the severe H1N1 and paraquat poisoning lung injury, argues for the possible action of Thal on COVID-19 induced lung effects and cytokine storm (9–12). Recent reviews on COVID-19 treatment endorse the possibility of usage of Thal and its analogs for treating COVID-19 patients. Thal, sadly known for its teratogenic action (13), has been shown to have potent immunomodulatory/anti-inflammatory activities (14), to prevent the development of bleomycin-induced pulmonary fibrosis in mice by blocking the TGF-β1-pathway (9), to improve respiratory symptoms and life quality in patients with IPF (10, 11) or H1N1 infection (12). Based on these observations, a repositioning study with Thal, in COVID-19 patients, has been performed in China with positive results (15). Moreover, Len at very high doses has been shown to induce ACE2 down-modulation through a post-translational mechanism (16). We have long studied the molecular basis of Thal’s teratogenic effects, specifically its role in degrading DNp63a and TAp63a proteins through the ubiquitin ligase CRL4 Cereblon (CRBN) (17). The p63 proteins are members of the p53-family of transcription factors and consist of ten isoforms with distinct and important biological roles during development (18, 19). In epithelial cells, the DNp63α isoform is the most prominent one regulating proliferation, apoptosis and differentiation (20). In addition, DNp63α regulates the transcription of several cytokines, such as IL-1a (21), IL-31, IL-33 (22) and IFN-g (23). Based on these observations, we reasoned that the beneficial effects upon Thal treatment observed in COVID–19 patients might be related to DNp63α degradation with subsequent modulation of its transcriptional targets. These targets would include already-known factors, such as inflammatory cytokines and novel targets such as the SARS-CoV-2 entry receptor ACE2. Materials and methods Cell culture, treatments and transfection . A431 (human epidermoid cell line; ATCC# CCL-1555) and A549 (human lung carcinoma cell line; ATCC# CCL-185) were kindly provided by Dr Ada Sacchi (Istituto Nazionale Tumori Regina Elena, Rome - Italy), H1299 (human lung carcinoma cell line; ATCC# CRL-5803) was kindly provided by Dr Giovanni Blandino (Istituto Nazionale Tumori Regina Elena, Rome - Italy), U-2 OS (human osteosarcoma cell line; ATCC# HTB-96) was kindly provided by Prof Francesco Blasi (Università degli Studi di Milano, Milan - Italy) and HaCaT (human keratinocyte cell line) was kindly provided by Prof. Antonio Costanzo (Humanitas Research Hospital, Milan -Italy). All cells were maintained in DMEM medium enriched with 10% Fetal Bovine Serum (Euroclone #ECS0165L), 1mM L-glutamine (Euroclone #ECB3000D), 100 units/mL penicillin and 100 µg/mL streptomycin (Euroclone #ECB3001D) at 37°C in a humidified atmosphere of 5% (v/v) CO 2 . Cells were maintained in culture for no more than ten passages and underwent routine testing to ensure they are mycoplasma-free. For treatments with Thalidomide (Tocris #50-35-1) and Lenalidomide (LGC products #191732-72-6), 5x10 4 cells were seeded onto 24-well multi-plates and 20 hrs later incubated with the different drug concentrations and times indicated in the figure legends. For transient transfection, 5x10 4 A431 cells were plated in 24 well multi-plates and on the next day transfected with Lipofectamine 2000 (Invitrogen #11668019) with increasing amount of CEREBLON (CRBN) encoding plasmid or with four different p63-Small Hairpin RNA vectors (sh-p63) with sequence homology to four different regions of p63 mRNA (OriGene Technologies # TF308688); shRNA-SCRAMBLED (sh-SCRB) was used as control. For U-2 OS, 5x10 4 cells were plated in 24 well multi-plates and the next day transfected with Lipofectamine 2000 with increasing amount of ΔNp63α encoding plasmid. Western blot (WB) analysis . At the indicated times, cells were lysed in 100 µl of Loading Buffer 2X [2% sodium dodecyl sulfate, 30% glycerol, 144 mM β-mercaptoethanol, 100 mM Tris–HCl pH 6.8 and 0.1% Bromo-Phenol Blue]. Samples were incubated at 98°C for 10 mins and resolved by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (Amersham #GEH10600001). The blots were incubated with the following antibodies (Abs): anti-p63 4A4 (Santa Cruz Biotechnology sc-8431), anti-ACE2 (Abcam ab15348), anti-CRBN (Cell Signaling Technology D8H3S), and anti-Actin (Santa Cruz Biotechnology sc-8432). The following secondary Abs were used: goat anti-mouse IgG-HRP (Santa Cruz Biotechnology sc-2005), goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology sc-2030). Proteins were visualized by an enhanced chemiluminescence method (Genespin #STSE500) according to manufacturer’s instructions using a Chemidoc touch (Biorad). Plasmids. The plasmids carrying ΔNp63α, CRBN, or shCRBN were previously described ( 17 ). RNA Purification, Reverse Transcriptase RT-PCR, and Quantitative Real-Time qPCR Analyses. Total mRNA from cells was isolated using the RNeasy miniKit (Qiagen #74104). cDNA was synthesized by M-MLV RTase and amplified with GoTaq DNA polymerase (Promega #M3001). For quantitative PCR analysis, mRNA expression level was evaluated using Power SYBR Green PCR master mix with ABI Prism 7500HT Fast Real-Time PCR System Detector (Applied Biosystems). Relative mRNA expression levels were determined by using the 2-∆∆CT method, employing GAPDH gene expression for data normalization. All reactions were performed in triplicate. Primer sequences are as follows: ACE2 Forward 5’-CAT TGG AGC AAG TGT TGG ATC TT-3’; ACE2 Reverse 5’-GAG CTA ATG CAT GCC ATT CTC A -3’; GAPDH Forward 5’-TCC CTG AGC TGA ACG GGA AG-3’; GAPDH Reverse 5’-GGA GGA GTG GGT GTC GCT GT-3’. ELISA assay. HaCaT cells, 1.8x10 5 , were plated in 35 mm culture dishes and treated with recombinant human TNF-α (rhTNF-α; Biolegend # BMS301) 5 ng/mL for 7 or 17 hrs or with 0.5 µg/mL LPS from E. coli ( SIGMA #O111:B4) for 4, 6, or 17 hrs. Quantification of IL-8 in the supernatants of treated HaCaT cells was performed by ELISA assay (hIL-8; ImmunoTools #31670089) according to the manufacturer’s instructions. The Elisa plates were read by microplate reader (SAFAS MP96). Chromatin Immunoprecipitation (ChIP). A431 cells, 4x10 6 , were plated in 15 cm culture dishes and treated with LPS 0.5 µg/mL for 2 hrs. Then, proteins were cross-linked to DNA in living nuclei and ChIP assay was performed using the MAGnify™ Chromatin Immunoprecipitation System (Thermo Fisher #492024), as described by the manufacturer. The following quantities of Abs were used to immunoprecipitate the DNA-protein complexes: 2 µg of anti-p63α (D2K8X) XP® Rabbit mAb (Cell Signaling Technology #131095), 2 µg of anti-acetylated H4-histone Ab (Biorad # AHP148) and 1 µg of an unrelated, negative control Ab. DNA fragments obtained by ChIP assay were analyzed by qPCR using specific primers spanning the RE found in the first intron of the ACE2 gene. Primers specific for exon 8 of the ACE2 gene were used as negative control. As a positive control for PCR and as normalizer, DNA prepared from samples prior to immunoprecipitation (whole cell lysates) was used as total or input DNA. Primer sequences are as follows: First intron of the ACE2 gene (+ 1067; +1087): Forward: 5’-ACGACGCGTGTGGAGAAGTCCATCAGA-3’; Reverse: 5’-ACGAGATCTG GTCA ACCACACATACCA-3’; Exon 8 ACE2 gene (+ 19634; +19730): Forward: 5’-GGGATGCACAGA GAATATTCAAGG-3’; Reverse: 5’-AGACTGCTTTCTGAACATTTCCTG-3’. In vitro SARS-CoV-2 spike protein pseudovirus infection. A baculovirus expressing the Green Fluorescent Protein (GFP) and pseudotyped with SARS-CoV-2 spike protein (Montana Molecular #C1110G) was employed to evaluate its entry into cells through ACE2 receptor. In particular, 5x10 3 A431 cells were plated in triplicates in 96-well plates. Thal was added 17 hrs post cell seeding; after 24 hrs of treatments, 50 uL of pseudo-SARS-CoV-2 suspension (viral titer: 2x10 10 Viral Genes (VG) per mL) were added following the manufacturer instructions. At 30 hrs post-infection the cells were washed with PBS and fixed with 4% formaldehyde in PBS for 10 mins at room temperature. Cells were incubated for 10 mins with reagent containing 4,6-DiAmidino-2-PhenylIndole (DAPI) and washed with PBS. Images were acquired using a Nikon CSU-W1 microscope in widefield mode, using a 20x objective and analyzed with FIJI ( 24 ). Results Thalidomide and Lenalidomide at clinically relevant concentrations reduce ACE2 expression . We first investigated whether Thal or Len could modulate the expression of ACE2 receptor, as a consequence of ΔNp63α degradation in HaCaT human keratinocytes and A431 human epidermoid cells, which express both ΔNp63α and ACE2 proteins. Cells were treated for 24 hrs with 10 or 100 µM Thal or 1 or 5 µM Len. As expected ( 17 ), ΔNp63α protein was degraded by the pharmacological treatments, with the ACE2 levels decreasing in parallel with the reduction of ΔNp63α levels in both cell lines (Fig. 1 A). We then verified whether Thal or Len could modulate ACE2 expression in p63-null cells in a dose-dependent manner, since it has been reported that Len at high concentrations induces ACE2 downmodulation by a posttranslational mechanism ( 16 ). At the highest concentrations of Thal, we did not observe ACE2 downmodulation in the p63-null U-2 OS human osteosarcoma cells. In contrast, the treatment with Len at the high concentrations reported to modulate ACE2 expression ( 16 ) ( i.e ., 80–100 µM) resulted to be toxic, as it can be inferred by the concomitant reduction of the actin levels (Fig. 1 B). Next, to investigate the modulation of ACE2 by ΔNp63α at the transcriptional level in response to Thal or Len treatment, we treated for 24 hrs with 100 µM Thal or 5 µM Len the following cells: A431, HaCaT and A549 cells, all expressing ΔNp63α and ACE2, and H1299 cells, not expressing any of the p63 isoforms. In ΔNp63α proficient cells, ΔNp63α protein was degraded by both treatments with a concomitant decrease of ACE2 mRNA levels suggesting that ACE2 might be a p63 transcriptional target, whereas, we did not observe ACE2 mRNA decrease in the p63-null cells (Fig. 1 C). Taken together, these results suggest that ACE2 could be a ΔNp63α target gene with Thal or Len leading to CRBN-mediated ΔNp63α degradation (CRBN is part of the E3 ubiquitin ligase complex that targets ΔNp63α for degradation upon Thal treatment ( 17 , 25 ) that in turn would give rise to reduced ACE2 transcription. Thalidomide reduce ACE2 expression through CRBN-mediated ΔNp63α degradation . To verify this hypothesis, we transiently transfected the p63-null U-2 OS cells with a ΔNp63α encoding plasmid and observed a positive correlation between expression levels of ACE2 and ΔNp63α (Fig. 2 A). Moreover, evaluation of ACE2 mRNA by qRT-PCR in parallel samples showed increased ACE2 mRNA levels in the samples transfected with the ΔNp63α plasmid, thus indicating that ACE2 might be a ΔNp63α transcriptional target (Fig. 2 B). This point was further reinforced by p63 silencing in HaCaT and A431 cells transfected with small hairpin RNA (shRNA) plasmids targeting the p63 mRNA. For this type of experiment, we used four different p63 shRNA vectors (OriGene) with sequence homology to four different regions of the p63 mRNA, with the sh-4 vector resulting in the strongest effect on ΔNp63α silencing and a concomitant decrease of ACE2 protein levels, in both cell lines (Fig. 2 C and 2 D). Finally, we reduced ΔNp63α protein levels by transfecting HaCaT and A431 cells with the CRBN encoding plasmid. As expected ( 17 ), CRBN overexpression led to a dose-dependent ΔNp63α degradation in both cell lines (Fig. 2 E) that was paralleled, also in this case, by ACE2 downmodulation, supporting the idea that ACE2 levels are directly correlated with ΔNp63α levels. ACE2 is a new ΔNp63α target gene. One of the main problems with SARS-CoV-2 infection is the triggering of a “cytokine storm” ( 26 ) a hyper-inflammatory state characterized by production of extremely high levels of proinflammatory cytokines that eventually leads to patient death. In order to mimic the hyper-inflammatory state in vitro , HaCaT cells were treated with LPS or the proinflammatory cytokine TNF-α, both known to stabilize ΔNp63α protein levels and to stimulate cytokine production, possibly by stabilized ΔNp63α acting on the promoter of several cytokine genes ( 27 – 29 ). Upon TNF-α and LPS treatments, both ΔNp63α and ACE2 expression levels were induced in dose-dependent and time-dependent manner (Fig. 3 A, lower panel). The levels of IL-8, known to be overproduced in COVID-19 patients and expressed in HaCaT cells ( 30 – 31 ) were also increased by our treatments (Fig. 3 A, upper panel), likely by stabilized ΔNp63α. We then verified whether p53-family Responsive Elements (RE) were present in the regulatory regions of the ACE2 gene. For this purpose, we queried the p53Fam-Tag database ( 32 ) and identified one strong putative p53/p63-RE composed by three decamers in the first intron of the ACE2 gene (Fig. 3 B). To evaluate the in vivo recruitment of ΔNp63α on the identified p53/p63-RE, a Chromatin ImmunoPrecipitation assay (ChIP) was performed. Cross-linked chromatin from A431 cells treated with LPS 0.5 µg/ml for 2 hrs was immunoprecipitated with anti-acetylated H4-histone or anti-p63α Abs. In the presence of LPS ( i.e. , 2 hrs treatment), but not in the untreated control cells, ΔNp63α was consistently recruited on the p53/p63-RE of the ACE2 gene (Fig. 3 D). The increased p63 occupancy was accompanied by an increase in histone H4 acetylation (Fig. 3 D) and, consistently, in ACE2 protein levels (Fig. 3 C). As negative control, the exon 8 of the ACE2 gene, not containing any p53/p63-RE, was not amplified in the same samples (Fig. 3 E). Taken together, these results clearly indicate that ACE2 is a new target gene of ΔNp63α. Thalidomide weakens in vitro infection by pseudo-SARS-CoV-2. It has been reported that COVID-19 patients treated with Thal had a faster recovery in respect to untreated patients ( 15 ). From the data obtained, we hypothesized that the observed protection might be due to diminished viral re-entry due to ACE2 downmodulation upon Thal treatment as a consequence of ΔNp63α degradation. To verify this hypothesis, we pretreated A431 cells with 100 µM Thal for 24 hrs before adding, for additional 24 hrs, the pseudo-SARS-CoV-2, a GFP-expressing baculovirus pseudotyped with the SARS-CoV-2 spike protein (Fig. 4 A). We found that Thal pretreatment impairs pseudoviral infection in vitro , as evidenced by reduced GFP signals in the Thal pretreated samples compared with the controls (Fig. 4 B and 4 C). Altogether, these data offer a mechanistic explanation of the protective effect from severe COVID-19 observed in patients treated with Thal ( 15 ). Discussion COVID-19 is an infectious disease caused by SARS-CoV-2 that started to spread at the end of 2019. The impellent need for effective therapies at the beginning of the pandemic drove several drug repositioning approaches to tackle the severe adverse effects of the virus infection ( 5 ). Among these, the usage of the immunomodulatory drug Thal was proposed, and its administration proved effective in fastening patient recovery and reducing the levels of inflammatory cytokines in the serum of the patients ( 15 ). Thal was originally used as anti-emetic compound to treat morning sickness of pregnant women and was withdrawn from the market in 1961 due to its teratogenic effects ( 6 , 13 ). This adverse effect was recently shown to depend on Thal-induced degradation of ΔNp63α and TAp63a, two isoforms of the p63 transcription factor known to have essential roles in skin and limb development ( 17 ). Nonetheless, this drug has found usage in clinical practice due to its anti-tumoral, immunomodulatory, and anti-inflammatory effects ( 7 , 33 ). In addition, it has been observed that SARS-CoV-2 virus induced lower mortality in Len-treated Multiple Myeloma patients compared to Len naïve counterparts ( 34 ), supporting that Thal and its analogues might be useful in the management of COVID-19 patients. However, the underlying molecular mechanism is still unclear. Starting from these evidences, we hypothesized that the beneficial effects observed in COVID-19 patients treated with Thal could stem from ΔNp63α degradation, with p63 eventually playing a role on the regulation of ACE2. Noteworthy, this p63-mediated mechanism of protection by SARS-CoV-2 infection has been observed in studies exploiting other known inducers of p63-degradation, such as metformin ( 35 – 36 ). Supporting this hypothesis, we identified in silico a p53/p63 RE in the ACE2 gene and demonstrated that, in conditions mimicking the well-known COVID-19 associated cytokine storm, p63 does indeed bind to this element. On top of this, ACE2 mRNA and protein levels both scale with p63 abundance in the cell, as shown by p63 overexpression or silencing further proving that p63 is indeed active at the ACE2 locus to promote ACE2 expression. We speculated that the effects observed in COVID-19 patients treated with Thal or Len could likely be caused by a p63-degradation dependent reduction of ACE2 levels, and that this downmodulation could reduce the SARS-CoV-2 viral infection and, in COVID-19 patients, viral re-entry. This hypothesis holds true as we show that Thal induces ACE2 reduction only in cells expressing p63 and reduces infection by a pseudo-SARS-CoV-2 virus. Overall, our work sheds new light onto current comprehension of Thal and p63 molecular details at various levels. First, our data envisage new roles of p63 on regulation of cell protein expression and support the general concept that Thal may efficiently modify the expression of genes through p63 regulation, among which is ACE2 . Second, they support a protective role for Thal against SARS-CoV-2 viral infection and explain the molecular details of how this compound exerts its functions, potentially making it a valuable option in the management of SARS-CoV-2 or other ACE2-dependent infections. Third, this work represents a proof of concept of a pharmacological substance that may play an effect on ACE2. This effect opens to new consideration on possible roles that the modulation of ACE2 cellular levels may offer in the clinical practice. Indeed, modulation of ACE2 levels with Thal may prevent or effectively contrast SARS-CoV-2 infection if properly given in early phases of the virus attack. While these data envisage a potential new preventive approach to SARS-CoV-2 also pose some important potential problems related to the use of Thal, the major one being the need to consider the negative effect of ACE2 blockage on the Renin-Angiotensin-Aldosterone System and specifically on hypertension and its effects on heart and renal function. It seems that specific anti-Angiotensin II therapies such as ARBs should follow and/or be given in concomitance with Thal. Clinical trials should be designed to resolve this aspect. Declarations Acknowledgements The authors would like to thank Maria Pia Gentileschi (Regina Elena National Cancer Institute) for technical support. Funding The present study was supported by Linea2, 2020-2021 (Università degli Studi di Milano) to L.G. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors' contributions LG, AT and SS conceived and designed the study. SP, LM, RQ, IV, AA, FM, and MP performed experiments and analyzed the data. LG, AT and SS wrote and revised the manuscript. LG, AT and SS confirm the authenticity of all the raw data. All authors read and approved the final manuscript. Ethics approval and consent to participate Not applicable. Patient consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, Müller MA, Drosten C, Pöhlmann S: SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181(2): 271-280, 2020. doi: 10.1016/j.cell.2020.02.052. Liu M, Wang T, Zhou Y, Zhao Y, Zhang Y, Li J: Potential Role of ACE2 in Coronavirus Disease 2019 (COVID-19) Prevention and Management. 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Si H, Lu H, Yang X, Mattox A, Jang M, Bian Y, Sano E, Viadiu H, Yan B, Yau C, Ng S, Lee SK, Romano RA, Davis S, Walker RL, Xiao W, Sun H, Wei L, Sinha S, Benz CC, Stuart JM, Meltzer PS, Van Waes C, Chen Z: TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB cREL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer. Oncogene 35(44): 5781-5794, 2016. doi: 10.1038/onc.2016.112. Liu X, Yin S, Chen Y, Wu Y, Zheng W, Dong H, Bai Y, Qin Y, Li J, Feng S, Zhao P: LPS‑induced proinflammatory cytokine expression in human airway epithelial cells and macrophages via NF‑κB, STAT3 or AP‑1 activation. Mol Med Rep 17(4): 5484-5491, 2018. doi: 10.3892/mmr.2018.8542. Choi DH, Hwang HS: Anti-inflammation activity of brazilin in TNF-α induced human psoriasis dermatitis skin model. Appl Biol Chem 62: 46, 2019. https://doi.org/10.1186/s13765-019-0455-z. Takada K, Komine-Aizawa S, Hirohata N, Trinh QD, Nishina A, Kimura H, Hayakawa S: Poly I:C induces collective migration of HaCaT keratinocytes via IL-8. BMC Immunol 18(1):19, 2017. doi: 10.1186/s12865-017-0202-3. Yang S, Jiang Y, Yu X, Zhu L, Wang L, Mao J, Wang M, Zhou N, Yang Z, Liu Y, Zhu T: Polyphyllin I Inhibits Propionibacterium acnes-Induced IL-8 Secretion in HaCaT Cells by Downregulating the CD36/NOX1/ROS/NLRP3/IL-1β Pathway. Evid Based Complement Alternat Med 2021: 1821220, 2021. doi: 10.1155/2021/1821220. Sbisà E, Catalano D, Grillo G, Licciulli F, Turi A, Liuni S, Pesole G, De Grassi A, Caratozzolo MF, D'Erchia AM, Navarro B, Tullo A, Saccone C, Gisel A: p53FamTaG: a database resource of human p53, p63 and p73 direct target genes combining in silico prediction and microarray data. BMC Bioinformatics 8 Suppl 1: S20, 2007. doi: 10.1186/1471-2105-8-S1-S20. Brigle K, Rogers B: Pathobiology and Diagnosis of Multiple Myeloma. Semin Oncol Nurs 33(3): 225-236, 2017. doi: 10.1016/j.soncn.2017.05.012. Tailor IK, Alshehry NF, Zaidi SZ, Marei MA, Motabi IH, Alfayez M, Altaf SY: Outcome of Myeloma Patients with COVID-19 on Active Lenalidomide-Based Therapy: Does Lenalidomide Protect From Severe COVID-19? Hematol Oncol Stem Cell Ther 16(1): 88-90, 2023. doi: 10.1016/j.hemonc.2020.08.002. Ojeda-Fernández L, Foresta A, Macaluso G, Colacioppo P, Tettamanti M, Zambon A, Genovese S, Fortino I, Leoni O, Roncaglioni MC, Baviera M: Metformin use is associated with a decrease in the risk of hospitalization and mortality in COVID-19 patients with diabetes: A population-based study in Lombardy. Diabetes Obes Metab 24(5): 891-898, 2022. doi: 10.1111/dom.14648. Yi Y, Chen D, Ao J, Sun S, Wu M, Li X, Bergholz J, Zhang Y, Xiao ZX: Metformin Promotes AMP-activated Protein Kinase-independent Suppression of ΔNp63α Protein Expression and Inhibits Cancer Cell Viability. J Biol Chem 292(13): 5253-5261, 2017. doi: 10.1074/jbc.M116.769141. Supplementary Files KEYMESSAGES.docx Cite Share Download PDF Status: Published Journal Publication published 18 Sep, 2024 Read the published version in Journal of Molecular Medicine → Version 1 posted Editorial decision: Major Revisions Needed 24 Apr, 2024 Reviewers agreed at journal 03 Apr, 2024 Reviewers invited by journal 03 Apr, 2024 Editor assigned by journal 27 Mar, 2024 First submitted to journal 26 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4162662","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":287239494,"identity":"390540f6-f5ad-41ea-9b03-e2294170f419","order_by":0,"name":"SARA POZZI","email":"","orcid":"","institution":"Università degli Studi di Milano: Universita degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"SARA","middleName":"","lastName":"POZZI","suffix":""},{"id":287239495,"identity":"b83211e8-a229-44a5-960a-958346116ae1","order_by":1,"name":"LAURA MONTEONOFRIO","email":"","orcid":"","institution":"Istituto Regina Elena","correspondingAuthor":false,"prefix":"","firstName":"LAURA","middleName":"","lastName":"MONTEONOFRIO","suffix":""},{"id":287239496,"identity":"c4d10632-a59d-4e50-a90a-ab02e8d6d487","order_by":2,"name":"ROBERTO QUADRI","email":"","orcid":"","institution":"Università degli Studi di Milano: Universita degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"ROBERTO","middleName":"","lastName":"QUADRI","suffix":""},{"id":287239497,"identity":"72180044-e52e-4957-b9bb-a850c752899d","order_by":3,"name":"ILARIA VIRDIA","email":"","orcid":"","institution":"Istituto Regina Elena","correspondingAuthor":false,"prefix":"","firstName":"ILARIA","middleName":"","lastName":"VIRDIA","suffix":""},{"id":287239498,"identity":"571a70b3-62d1-4e7e-824b-e5eebf679e57","order_by":4,"name":"ALESSANDRA AMENDOLARE","email":"","orcid":"","institution":"IBIOM CNR: Istituto di Biomembrane Bioenergetica e Biotecnologie Molecolari Consiglio Nazionale delle Ricerche","correspondingAuthor":false,"prefix":"","firstName":"ALESSANDRA","middleName":"","lastName":"AMENDOLARE","suffix":""},{"id":287239499,"identity":"d64ea1bd-f8eb-44c6-8add-caae78863db2","order_by":5,"name":"FLAVIANA MARZANO","email":"","orcid":"","institution":"IBBE: Istituto di Biomembrane Bioenergetica e Biotecnologie Molecolari Consiglio Nazionale delle Ricerche","correspondingAuthor":false,"prefix":"","firstName":"FLAVIANA","middleName":"","lastName":"MARZANO","suffix":""},{"id":287239500,"identity":"b0f895b1-e4f3-443c-bc71-8594bdff229f","order_by":6,"name":"MOIRA PARONI","email":"","orcid":"","institution":"Università degli Studi di Milano: Universita degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"MOIRA","middleName":"","lastName":"PARONI","suffix":""},{"id":287239501,"identity":"e3e94eec-b82d-45e0-b593-101d95ff3111","order_by":7,"name":"APOLLONIA TULLO","email":"","orcid":"","institution":"IBIOM CNR: Istituto di Biomembrane Bioenergetica e Biotecnologie Molecolari Consiglio Nazionale delle Ricerche","correspondingAuthor":false,"prefix":"","firstName":"APOLLONIA","middleName":"","lastName":"TULLO","suffix":""},{"id":287239502,"identity":"20b7812d-054b-4d28-b202-90c687ec5a68","order_by":8,"name":"SILVIA SODDU","email":"","orcid":"","institution":"Regina Elena Institute: Istituto Regina Elena","correspondingAuthor":false,"prefix":"","firstName":"SILVIA","middleName":"","lastName":"SODDU","suffix":""},{"id":287239503,"identity":"d7c9769d-5004-433a-9884-e852a2c2c698","order_by":9,"name":"LUISA GUERRINI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBACPgbGBjjnAIOBDQMDM5iBWwsbmpY0mBbcetjQ+IdhDDxaJJKbP/xgqE3slz778HBBwfnE7ezciQcYCv7g0ZLYJtnDcDxxZl+6weEZBrcTdzbzbsDvMKAWBh6GY4kbzrAxHOYBatlwmLCW5o9/gFr2Q7ScI0pLgzQPQ03iBh6wlgNEaOF52CYtY3DAeAbElmRjsJYEA2OcWvjZ0x9/fFNRJ9vfw8b8meePneyG82c3f/jwRw6nFggwOIwmkEBAAxDUEVYyCkbBKBgFIxcAAIFyUxsSRPHTAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5316-4699","institution":"Università degli Studi di Milano: Universita degli Studi di Milano","correspondingAuthor":true,"prefix":"","firstName":"LUISA","middleName":"","lastName":"GUERRINI","suffix":""}],"badges":[],"createdAt":"2024-03-25 10:47:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4162662/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4162662/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00109-024-02485-x","type":"published","date":"2024-09-18T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54316317,"identity":"b834e7db-6127-465d-9191-8a542d576dd2","added_by":"auto","created_at":"2024-04-08 17:50:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":97062,"visible":true,"origin":"","legend":"\u003cp\u003eThal and Len at clinically relevant concentrations reduce ACE2 expression. (A) HaCaT and A431 cells were treated for 24 hrs with Len (1 or 5 µM) or Thal (10 or 100 µM). Cell extracts were prepared and analyzed by WB with anti-ACE2 or anti-p63 Abs. Actin was used as loading control and one representative experiment is shown. (B) p63-null U-2 OS cells were treated for 24 hrs with increasing concentrations of Len (1, 5, 15, 50, 80, 100 µM) or Thal (10, 50, 100 µM). Cells extracts were analyzed by WB with anti-ACE2 Ab. Actin was used as loading control and one representative experiment is shown. (C) A431, A549, HaCaT and H1299 cells were treated with Thal 100 µM or Len 5 µM for 8 hrs; DMSO was used as control (NT). Lower panel: cell extracts were prepared and analyzed by WB with anti-p63 Ab; GAPDH was used as loading control and one representative experiment is shown. Upper panel: compared to the relative Ctrl, ACE2 mRNA levels decreased in p63-proficient A431, A459 and HaCaT cells treated with Thal or Len but not in p63-deficient H1299 cells. ACE2 mRNA was evaluated by qRT-PCR and mRNA levels were normalized using GAPDH and the means ± standard errors (SE) of three replicates are shown.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4162662/v1/22a972a498c522b08556be19.png"},{"id":54315165,"identity":"cb754be1-738f-4345-a1f2-466c1c90c7aa","added_by":"auto","created_at":"2024-04-08 17:42:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":141835,"visible":true,"origin":"","legend":"\u003cp\u003eΔNp63α and ACE2 parallel modulation at both protein and mRNA levels. (A) p63-null U-2 OS cells were transiently transfected with 10 and 25 ng of ΔNp63α expression vector. After 24 hrs from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2 or anti-p63 Abs. Actin was used as loading control and one representative experiment is shown. (B) ACE2 mRNA levels were evaluated by qRT-PCR and mRNA levels were normalized using GAPDH as housekeeping gene. Means ± SE of three replicates are shown. (C) HaCaT and A431 cells were transiently transfected with 100 ng of small hairpin RNA (shRNA) plasmids targeting p63 mRNA; four different p63 shRNA vectors with sequence homology to four different regions of p63 mRNA were used (#1, #2, #3, #4); the cells were also transfected with a control (100 ng), shRNA-SCR, that encodes for a RNA that is not complementary to any mRNA sequence. After 24 hrs from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2 and anti-p63 Abs. Actin was used as loading control one representative experiment is shown. (D) A431 cells were transiently transfected with 100, 250, and 500 ng of the shRNA#4; cells were also transfected with a control shRNA-SCR (500 ng). After 24 hrs from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2 and anti-p63 Abs. Actin was used as loading control one representative experiment is shown. (E) HaCaT and A431 cells were transiently transfected with 100 and 250 ng of CRBN encoding plasmid. After 24 hrs from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2, anti-p63, and anti-CRBN Abs. Actin was used as loading control one representative experiment is shown. Two bands of the CRBN protein are evident: ° stays for endogenous CRBN, * for transfected CRBN that has higher molecular weight due to an HA tag.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4162662/v1/73d64ed61ef31b43c345dbc5.png"},{"id":54315167,"identity":"30f33c1c-611f-424c-aabc-0988e0bae41e","added_by":"auto","created_at":"2024-04-08 17:42:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92759,"visible":true,"origin":"","legend":"\u003cp\u003eACE2 is a new ΔNp63α target gene. (A) HaCaT cells were treated with 5 ng/mL of TNF-α or with 0.5 µg/mL of LPS for the indicated times. Cell extracts were prepared and analyzed by WB with an anti-ACE2 and anti-p63 Abs. Actin was used as loading control and one representative experiment is shown. The cell supernatants were collected and IL-8 levels quantified by ELISA assay. (B) Schematic representation and sequence of the p53/p63RE identified in the first intron of \u003cem\u003eACE2. \u003c/em\u003e(C)\u003cstrong\u003e \u003c/strong\u003eA431\u003cstrong\u003e \u003c/strong\u003ecells were treated or not with LPS 0.5 mg/ml for 2 hrs. Cell extracts were prepared and analyzed by WB using anti-ACE2 and anti-p63 Abs. Actin was used as loading control and one representative experiment is shown. (D-E\u003cstrong\u003e)\u003c/strong\u003e. A431\u003cstrong\u003e \u003c/strong\u003ecells were treated with LPS for 2 hrs and then collected for Chromatin Immunoprecipitation (ChIP) analysis.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4162662/v1/83c5ccb22f51a15f704dee82.png"},{"id":54315166,"identity":"243e8287-5a68-461d-ac3b-00f55299073d","added_by":"auto","created_at":"2024-04-08 17:42:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":961640,"visible":true,"origin":"","legend":"\u003cp\u003eThal impairs \u003cem\u003ein vitro\u003c/em\u003e infection by GFP-expressing pseudo-SARS-CoV-2. (A) Schematic representation of the experiment shown in B. (B) Immunofluorescent staining of A431 cells treated with Thal 100 µM; after 24 hrs treatment, 50 µL of pseudo-SARS-CoV-2 suspensions were added to the cells following the manufacturer instructions; the viral titer was 2x10\u003csup\u003e10\u003c/sup\u003e Viral Genes (VG) per mL. After 24 hrs post-infection, cells were washed with PBS and fixed with 4% formaldehyde in 1X PBS for 10 mins at room temperature. Nuclei were stained with DAPI. Images were acquired using a Nikon CSU-W1 microscope in widefield mode, using a 20x objective and analyzed with FIJI and representative images are shown. Scale bar 150 mm. (C) Quantification of GFP reduction in Thal pretreated samples.\u003c/p\u003e\n\u003cp\u003e[24]\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4162662/v1/e815066eaf6f5b5dff7b0e6f.png"},{"id":65104027,"identity":"073502b9-7d5f-4073-b32d-7ab209cb7ba3","added_by":"auto","created_at":"2024-09-23 16:10:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1935765,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4162662/v1/e67a7adf-6634-4e4b-bd4f-1419e6738ce6.pdf"},{"id":54315163,"identity":"8d91d744-d731-434a-99fb-f603b643ee41","added_by":"auto","created_at":"2024-04-08 17:42:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14010,"visible":true,"origin":"","legend":"","description":"","filename":"KEYMESSAGES.docx","url":"https://assets-eu.researchsquare.com/files/rs-4162662/v1/045463626f524ac6fc249590.docx"}],"financialInterests":"","formattedTitle":"Molecular mechanisms of Thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSARS-CoV-2 infection, mediated by the binding of the viral Spike (S) protein to the ACE2 receptor in the host cells (1\u0026ndash;3) causes the COVID-19 disease. One of the associated complications is a cytokine storm which involves an uncontrolled release of cytokines and chemokines, leading to a systemic inflammatory response linked to lung failure and acute respiratory distress syndrome (ARDS). Given the time and cost involved in developing new therapies, repurposing existing drugs with known profiles has become a cost-effective strategy (4). Therefore, reassessing the efficacy of licensed and experimental drugs has been a go-on choice by the World Health Organization (WHO) and other health agencies to treat emerging health problems. With the initial lack of vaccines and effective agents against SARS-CoV-2, as well as public health emergencies, WHO had identified promising reposition therapies, such as the combination of two anti-HIV drugs (\u003cem\u003ei.e.,\u003c/em\u003e lopinavir and ritonavir) or the experimental antiviral compound, remdesivir (5). Furthermore, Thalidomide (Thal),\u0026nbsp;a small molecule drug with many years of history known to cause misery,\u0026nbsp;became a game changer for its multifaceted pharmacological effects such as immunomodulation, anti-inflammation, anti-angiogenesis, and anti-viral effects (6, 7). Several Thal derivatives have been designed, known as Immuno Modulatory drugs (IMids), among which the most used are Lenalidomide (Len) and Pomalidomide, that are approved by FDA for the treatment of several severe diseases like Multiple Myeloma, myelodysplastic syndrome (5q-), mantle cell lymphoma, and follicular lymphoma (8). IMids retain the anti-angiogenic and anti-inflammatory properties of Thal but are devoid of Thal teratogenic activity (6, 8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExtensive information available on Thal\u0026rsquo;s mechanisms of action, its efficacy and safety in the haemophagocytic syndrome-induced cytokine storm\u0026nbsp;and Idiopathic Pulmonary Fibrosis (IPF) and in the severe H1N1 and paraquat poisoning lung injury,\u0026nbsp;argues for the possible action of Thal on COVID-19 induced lung effects and cytokine storm (9\u0026ndash;12). Recent\u0026nbsp;reviews on COVID-19 treatment endorse the possibility of usage of Thal and its analogs for treating COVID-19 patients. Thal, sadly known for its teratogenic action (13), has been shown to have potent immunomodulatory/anti-inflammatory activities (14), to prevent the development of bleomycin-induced pulmonary fibrosis in mice by blocking the TGF-\u0026beta;1-pathway (9), to improve respiratory symptoms and life quality in patients with IPF (10, 11) or H1N1 infection (12). Based on these observations, a repositioning study with Thal, in COVID-19 patients, has been performed in China with positive results (15). Moreover, Len at very high doses has been shown to induce ACE2 down-modulation through a post-translational mechanism (16).\u003c/p\u003e\n\u003cp\u003eWe have long studied the molecular basis of Thal\u0026rsquo;s teratogenic effects, specifically its role in degrading \u0026nbsp;DNp63a and TAp63a proteins through the ubiquitin ligase CRL4 Cereblon (CRBN) (17). The p63 proteins are members of the p53-family of transcription factors and consist of ten isoforms with distinct and important biological roles during development (18, 19). In epithelial cells, the DNp63\u0026alpha; isoform is the most prominent one regulating proliferation, apoptosis and differentiation (20). In addition, DNp63\u0026alpha; regulates the transcription of several cytokines, such as IL-1a (21), IL-31, IL-33 (22) and IFN-g (23). Based on these observations, we reasoned that the beneficial effects upon Thal treatment observed in COVID\u0026ndash;19 patients might be related to DNp63\u0026alpha; degradation with subsequent modulation of its transcriptional targets. These targets would include already-known factors, such as inflammatory cytokines and novel targets such as the SARS-CoV-2 entry receptor ACE2.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cem\u003eCell culture, treatments and transfection\u003c/em\u003e. A431 (human epidermoid cell line; ATCC# CCL-1555) and A549 (human lung carcinoma cell line; ATCC# CCL-185) were kindly provided by Dr Ada Sacchi (Istituto Nazionale Tumori Regina Elena, Rome - Italy), H1299 (human lung carcinoma cell line; ATCC# CRL-5803) was kindly provided by Dr Giovanni Blandino (Istituto Nazionale Tumori Regina Elena, Rome - Italy), U-2 OS (human osteosarcoma cell line; ATCC# HTB-96) was kindly provided by Prof Francesco Blasi (Universit\u0026agrave; degli Studi di Milano, Milan - Italy) and HaCaT (human keratinocyte cell line) was kindly provided by Prof. Antonio Costanzo (Humanitas Research Hospital, Milan -Italy). All cells were maintained in DMEM medium enriched with 10% Fetal Bovine Serum (Euroclone #ECS0165L), 1mM L-glutamine (Euroclone #ECB3000D), 100 units/mL penicillin and 100 \u0026micro;g/mL streptomycin (Euroclone #ECB3001D) at 37\u0026deg;C in a humidified atmosphere of 5% (v/v) CO\u003csup\u003e2\u003c/sup\u003e. Cells were maintained in culture for no more than ten passages and underwent routine testing to ensure they are mycoplasma-free. For treatments with Thalidomide (Tocris #50-35-1) and Lenalidomide (LGC products #191732-72-6), 5x10\u003csup\u003e4\u003c/sup\u003e cells were seeded onto 24-well multi-plates and 20 hrs later incubated with the different drug concentrations and times indicated in the figure legends. For transient transfection, 5x10\u003csup\u003e4\u003c/sup\u003e A431 cells were plated in 24 well multi-plates and on the next day transfected with Lipofectamine 2000 (Invitrogen #11668019) with increasing amount of CEREBLON (CRBN) encoding plasmid or with four different p63-Small Hairpin RNA vectors (sh-p63) with sequence homology to four different regions of p63 mRNA (OriGene Technologies # TF308688); shRNA-SCRAMBLED (sh-SCRB) was used as control. For U-2 OS, 5x10\u003csup\u003e4\u003c/sup\u003e cells were plated in 24 well multi-plates and the next day transfected with Lipofectamine 2000 with increasing amount of ΔNp63α encoding plasmid.\u003c/p\u003e \u003cp\u003e \u003cem\u003eWestern blot (WB) analysis\u003c/em\u003e. At the indicated times, cells were lysed in 100 \u0026micro;l of Loading Buffer 2X [2% sodium dodecyl sulfate, 30% glycerol, 144 mM β-mercaptoethanol, 100 mM Tris\u0026ndash;HCl pH 6.8 and 0.1% Bromo-Phenol Blue]. Samples were incubated at 98\u0026deg;C for 10 mins and resolved by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (Amersham #GEH10600001). The blots were incubated with the following antibodies (Abs): anti-p63 4A4 (Santa Cruz Biotechnology sc-8431), anti-ACE2 (Abcam ab15348), anti-CRBN (Cell Signaling Technology D8H3S), and anti-Actin (Santa Cruz Biotechnology sc-8432). The following secondary Abs were used: goat anti-mouse IgG-HRP (Santa Cruz Biotechnology sc-2005), goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology sc-2030). Proteins were visualized by an enhanced chemiluminescence method (Genespin #STSE500) according to manufacturer\u0026rsquo;s instructions using a Chemidoc touch (Biorad).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePlasmids.\u003c/em\u003e The plasmids carrying ΔNp63α, CRBN, or shCRBN were previously described (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eRNA Purification, Reverse Transcriptase RT-PCR, and Quantitative Real-Time qPCR Analyses.\u003c/em\u003e Total mRNA from cells was isolated using the RNeasy miniKit (Qiagen #74104). cDNA was synthesized by M-MLV RTase and amplified with GoTaq DNA polymerase (Promega #M3001). For quantitative PCR analysis, mRNA expression level was evaluated using Power SYBR Green PCR master mix with ABI Prism 7500HT Fast Real-Time PCR System Detector (Applied Biosystems). Relative mRNA expression levels were determined by using the 2-∆∆CT method, employing GAPDH gene expression for data normalization. All reactions were performed in triplicate. Primer sequences are as follows: ACE2 Forward 5\u0026rsquo;-CAT TGG AGC AAG TGT TGG ATC TT-3\u0026rsquo;; ACE2 Reverse 5\u0026rsquo;-GAG CTA ATG CAT GCC ATT CTC A -3\u0026rsquo;; GAPDH Forward 5\u0026rsquo;-TCC CTG AGC TGA ACG GGA AG-3\u0026rsquo;; GAPDH Reverse 5\u0026rsquo;-GGA GGA GTG GGT GTC GCT GT-3\u0026rsquo;.\u003c/p\u003e \u003cp\u003e \u003cem\u003eELISA assay.\u003c/em\u003e HaCaT cells, 1.8x10\u003csup\u003e5\u003c/sup\u003e, were plated in 35 mm culture dishes and treated with recombinant human TNF-α (rhTNF-α; Biolegend # BMS301) 5 ng/mL for 7 or 17 hrs or with 0.5 \u0026micro;g/mL LPS from \u003cem\u003eE. coli\u003c/em\u003e ( SIGMA #O111:B4) for 4, 6, or 17 hrs. Quantification of IL-8 in the supernatants of treated HaCaT cells was performed by ELISA assay (hIL-8; ImmunoTools #31670089) according to the manufacturer\u0026rsquo;s instructions. The Elisa plates were read by microplate reader (SAFAS MP96).\u003c/p\u003e \u003cp\u003e \u003cem\u003eChromatin Immunoprecipitation (ChIP).\u003c/em\u003e A431 cells, 4x10\u003csup\u003e6\u003c/sup\u003e, were plated in 15 cm culture dishes and treated with LPS 0.5 \u0026micro;g/mL for 2 hrs. Then, proteins were cross-linked to DNA in living nuclei and ChIP assay was performed using the MAGnify\u0026trade; Chromatin Immunoprecipitation System (Thermo Fisher #492024), as described by the manufacturer. The following quantities of Abs were used to immunoprecipitate the DNA-protein complexes: 2 \u0026micro;g of anti-p63α (D2K8X) XP\u0026reg; Rabbit mAb (Cell Signaling Technology #131095), 2 \u0026micro;g of anti-acetylated H4-histone Ab (Biorad # AHP148) and 1 \u0026micro;g of an unrelated, negative control Ab. DNA fragments obtained by ChIP assay were analyzed by qPCR using specific primers spanning the RE found in the first intron of the \u003cem\u003eACE2\u003c/em\u003e gene. Primers specific for exon 8 of the \u003cem\u003eACE2\u003c/em\u003e gene were used as negative control. As a positive control for PCR and as normalizer, DNA prepared from samples prior to immunoprecipitation (whole cell lysates) was used as total or input DNA. Primer sequences are as follows: First intron of the \u003cem\u003eACE2\u003c/em\u003e gene (+\u0026thinsp;1067; +1087): Forward: 5\u0026rsquo;-ACGACGCGTGTGGAGAAGTCCATCAGA-3\u0026rsquo;; Reverse: 5\u0026rsquo;-ACGAGATCTG GTCA\u003c/p\u003e \u003cp\u003eACCACACATACCA-3\u0026rsquo;; Exon 8 \u003cem\u003eACE2\u003c/em\u003e gene (+\u0026thinsp;19634; +19730): Forward: 5\u0026rsquo;-GGGATGCACAGA GAATATTCAAGG-3\u0026rsquo;; Reverse: 5\u0026rsquo;-AGACTGCTTTCTGAACATTTCCTG-3\u0026rsquo;.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro SARS-CoV-2 spike protein pseudovirus infection.\u003c/em\u003e A baculovirus expressing the Green Fluorescent Protein (GFP) and pseudotyped with SARS-CoV-2 spike protein (Montana Molecular #C1110G) was employed to evaluate its entry into cells through ACE2 receptor. In particular, 5x10\u003csup\u003e3\u003c/sup\u003e A431 cells were plated in triplicates in 96-well plates. Thal was added 17 hrs post cell seeding; after 24 hrs of treatments, 50 uL of pseudo-SARS-CoV-2 suspension (viral titer: 2x10\u003csup\u003e10\u003c/sup\u003e Viral Genes (VG) per mL) were added following the manufacturer instructions. At 30 hrs post-infection the cells were washed with PBS and fixed with 4% formaldehyde in PBS for 10 mins at room temperature. Cells were incubated for 10 mins with reagent containing 4,6-DiAmidino-2-PhenylIndole (DAPI) and washed with PBS. Images were acquired using a Nikon CSU-W1 microscope in widefield mode, using a 20x objective and analyzed with FIJI (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eThalidomide and Lenalidomide at clinically relevant concentrations reduce ACE2 expression\u003c/em\u003e. We first investigated whether Thal or Len could modulate the expression of ACE2 receptor, as a consequence of ΔNp63α degradation in HaCaT human keratinocytes and A431 human epidermoid cells, which express both ΔNp63α and ACE2 proteins. Cells were treated for 24 hrs with 10 or 100 \u0026micro;M Thal or 1 or 5 \u0026micro;M Len. As expected (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), ΔNp63α protein was degraded by the pharmacological treatments, with the ACE2 levels decreasing in parallel with the reduction of ΔNp63α levels in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We then verified whether Thal or Len could modulate ACE2 expression in p63-null cells in a dose-dependent manner, since it has been reported that Len at high concentrations induces ACE2 downmodulation by a posttranslational mechanism (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). At the highest concentrations of Thal, we did not observe ACE2 downmodulation in the p63-null U-2 OS human osteosarcoma cells. In contrast, the treatment with Len at the high concentrations reported to modulate ACE2 expression (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) (\u003cem\u003ei.e\u003c/em\u003e., 80\u0026ndash;100 \u0026micro;M) resulted to be toxic, as it can be inferred by the concomitant reduction of the actin levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Next, to investigate the modulation of ACE2 by ΔNp63α at the transcriptional level in response to Thal or Len treatment, we treated for 24 hrs with 100 \u0026micro;M Thal or 5 \u0026micro;M Len the following cells: A431, HaCaT and A549 cells, all expressing ΔNp63α and ACE2, and H1299 cells, not expressing any of the p63 isoforms. In ΔNp63α proficient cells, ΔNp63α protein was degraded by both treatments with a concomitant decrease of ACE2 mRNA levels suggesting that ACE2 might be a p63 transcriptional target, whereas, we did not observe ACE2 mRNA decrease in the p63-null cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, these results suggest that ACE2 could be a ΔNp63α target gene with Thal or Len leading to CRBN-mediated ΔNp63α degradation (CRBN is part of the E3 ubiquitin ligase complex that targets ΔNp63α for degradation upon Thal treatment (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) that in turn would give rise to reduced ACE2 transcription.\u003c/p\u003e \u003cp\u003e \u003cem\u003eThalidomide reduce ACE2 expression through CRBN-mediated ΔNp63α degradation\u003c/em\u003e. To verify this hypothesis, we transiently transfected the p63-null U-2 OS cells with a ΔNp63α encoding plasmid and observed a positive correlation between expression levels of ACE2 and ΔNp63α (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Moreover, evaluation of ACE2 mRNA by qRT-PCR in parallel samples showed increased ACE2 mRNA levels in the samples transfected with the ΔNp63α plasmid, thus indicating that ACE2 might be a ΔNp63α transcriptional target (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This point was further reinforced by p63 silencing in HaCaT and A431 cells transfected with small hairpin RNA (shRNA) plasmids targeting the p63 mRNA. For this type of experiment, we used four different p63 shRNA vectors (OriGene) with sequence homology to four different regions of the p63 mRNA, with the sh-4 vector resulting in the strongest effect on ΔNp63α silencing and a concomitant decrease of ACE2 protein levels, in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Finally, we reduced ΔNp63α protein levels by transfecting HaCaT and A431 cells with the CRBN encoding plasmid. As expected (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), CRBN overexpression led to a dose-dependent ΔNp63α degradation in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) that was paralleled, also in this case, by ACE2 downmodulation, supporting the idea that ACE2 levels are directly correlated with ΔNp63α levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eACE2 is a new ΔNp63α target gene.\u003c/em\u003e One of the main problems with SARS-CoV-2 infection is the triggering of a \u0026ldquo;cytokine storm\u0026rdquo; (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) a hyper-inflammatory state characterized by production of extremely high levels of proinflammatory cytokines that eventually leads to patient death. In order to mimic the hyper-inflammatory state \u003cem\u003ein vitro\u003c/em\u003e, HaCaT cells were treated with LPS or the proinflammatory cytokine TNF-α, both known to stabilize ΔNp63α protein levels and to stimulate cytokine production, possibly by stabilized ΔNp63α acting on the promoter of several cytokine genes (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Upon TNF-α and LPS treatments, both ΔNp63α and ACE2 expression levels were induced in dose-dependent and time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, lower panel). The levels of IL-8, known to be overproduced in COVID-19 patients and expressed in HaCaT cells (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) were also increased by our treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, upper panel), likely by stabilized ΔNp63α.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then verified whether p53-family Responsive Elements (RE) were present in the regulatory regions of the \u003cem\u003eACE2\u003c/em\u003e gene. For this purpose, we queried the p53Fam-Tag database (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) and identified one strong putative p53/p63-RE composed by three decamers in the first intron of the \u003cem\u003eACE2\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To evaluate the \u003cem\u003ein vivo\u003c/em\u003e recruitment of ΔNp63α on the identified p53/p63-RE, a Chromatin ImmunoPrecipitation assay (ChIP) was performed. Cross-linked chromatin from A431 cells treated with LPS 0.5 \u0026micro;g/ml for 2 hrs was immunoprecipitated with anti-acetylated H4-histone or anti-p63α Abs. In the presence of LPS (\u003cem\u003ei.e.\u003c/em\u003e, 2 hrs treatment), but not in the untreated control cells, ΔNp63α was consistently recruited on the p53/p63-RE of the \u003cem\u003eACE2\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The increased p63 occupancy was accompanied by an increase in histone H4 acetylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and, consistently, in ACE2 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). As negative control, the exon 8 of the \u003cem\u003eACE2\u003c/em\u003e gene, not containing any p53/p63-RE, was not amplified in the same samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Taken together, these results clearly indicate that ACE2 is a new target gene of ΔNp63α.\u003c/p\u003e \u003cp\u003e \u003cem\u003eThalidomide weakens in vitro infection by pseudo-SARS-CoV-2.\u003c/em\u003e It has been reported that COVID-19 patients treated with Thal had a faster recovery in respect to untreated patients (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). From the data obtained, we hypothesized that the observed protection might be due to diminished viral re-entry due to ACE2 downmodulation upon Thal treatment as a consequence of ΔNp63α degradation. To verify this hypothesis, we pretreated A431 cells with 100 \u0026micro;M Thal for 24 hrs before adding, for additional 24 hrs, the pseudo-SARS-CoV-2, a GFP-expressing baculovirus pseudotyped with the SARS-CoV-2 spike protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We found that Thal pretreatment impairs pseudoviral infection \u003cem\u003ein vitro\u003c/em\u003e, as evidenced by reduced GFP signals in the Thal pretreated samples compared with the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAltogether, these data offer a mechanistic explanation of the protective effect from severe COVID-19 observed in patients treated with Thal (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCOVID-19 is an infectious disease caused by SARS-CoV-2 that started to spread at the end of 2019. The impellent need for effective therapies at the beginning of the pandemic drove several drug repositioning approaches to tackle the severe adverse effects of the virus infection (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Among these, the usage of the immunomodulatory drug Thal was proposed, and its administration proved effective in fastening patient recovery and reducing the levels of inflammatory cytokines in the serum of the patients (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThal was originally used as anti-emetic compound to treat morning sickness of pregnant women and was withdrawn from the market in 1961 due to its teratogenic effects (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This adverse effect was recently shown to depend on Thal-induced degradation of ΔNp63α and TAp63a, two isoforms of the p63 transcription factor known to have essential roles in skin and limb development (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Nonetheless, this drug has found usage in clinical practice due to its anti-tumoral, immunomodulatory, and anti-inflammatory effects (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In addition, it has been observed that SARS-CoV-2 virus induced lower mortality in Len-treated Multiple Myeloma patients compared to Len na\u0026iuml;ve counterparts (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), supporting that Thal and its analogues might be useful in the management of COVID-19 patients. However, the underlying molecular mechanism is still unclear. Starting from these evidences, we hypothesized that the beneficial effects observed in COVID-19 patients treated with Thal could stem from ΔNp63α degradation, with p63 eventually playing a role on the regulation of ACE2. Noteworthy, this p63-mediated mechanism of protection by SARS-CoV-2 infection has been observed in studies exploiting other known inducers of p63-degradation, such as metformin (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Supporting this hypothesis, we identified \u003cem\u003ein silico\u003c/em\u003e a p53/p63 RE in the \u003cem\u003eACE2\u003c/em\u003e gene and demonstrated that, in conditions mimicking the well-known COVID-19 associated cytokine storm, p63 does indeed bind to this element. On top of this, \u003cem\u003eACE2\u003c/em\u003e mRNA and protein levels both scale with p63 abundance in the cell, as shown by p63 overexpression or silencing further proving that p63 is indeed active at the \u003cem\u003eACE2\u003c/em\u003e locus to promote ACE2 expression. We speculated that the effects observed in COVID-19 patients treated with Thal or Len could likely be caused by a p63-degradation dependent reduction of ACE2 levels, and that this downmodulation could reduce the SARS-CoV-2 viral infection and, in COVID-19 patients, viral re-entry. This hypothesis holds true as we show that Thal induces ACE2 reduction only in cells expressing p63 and reduces infection by a pseudo-SARS-CoV-2 virus.\u003c/p\u003e \u003cp\u003eOverall, our work sheds new light onto current comprehension of Thal and p63 molecular details at various levels. First, our data envisage new roles of p63 on regulation of cell protein expression and support the general concept that Thal may efficiently modify the expression of genes through p63 regulation, among which is \u003cem\u003eACE2\u003c/em\u003e. Second, they support a protective role for Thal against SARS-CoV-2 viral infection and explain the molecular details of how this compound exerts its functions, potentially making it a valuable option in the management of SARS-CoV-2 or other ACE2-dependent infections. Third, this work represents a proof of concept of a pharmacological substance that may play an effect on ACE2. This effect opens to new consideration on possible roles that the modulation of ACE2 cellular levels may offer in the clinical practice. Indeed, modulation of ACE2 levels with Thal may prevent or effectively contrast SARS-CoV-2 infection if properly given in early phases of the virus attack. While these data envisage a potential new preventive approach to SARS-CoV-2 also pose some important potential problems related to the use of Thal, the major one being the need to consider the negative effect of ACE2 blockage on the Renin-Angiotensin-Aldosterone System and specifically on hypertension and its effects on heart and renal function. It seems that specific anti-Angiotensin II therapies such as ARBs should follow and/or be given in concomitance with Thal. Clinical trials should be designed to resolve this aspect.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Maria Pia Gentileschi (Regina Elena National Cancer Institute) for technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was supported by Linea2, 2020-2021 (Universit\u0026agrave; degli Studi di Milano) to L.G.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLG, AT and SS conceived and designed the study. SP, LM, RQ, IV, AA, FM, and MP performed experiments and analyzed the data. LG, AT and SS wrote and revised the manuscript. LG, AT and SS confirm the authenticity of all the raw data. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHoffmann M, Kleine-Weber H, Schroeder S, Kr\u0026uuml;ger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, M\u0026uuml;ller MA, Drosten C, P\u0026ouml;hlmann S: SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. 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The identification of novel approaches to tackle this step is instrumental for the development of therapies for the management of COVID-19 and other diseases with a similar mechanism of infection. Thalidomide, a drug sadly known for its teratogenic effects, has potent immunomodulatory and anti-inflammatory properties. Treatment with this drug has been shown to improve the immune functions of COVID-19 patients and proposed for the management of COVID-19 in clinical practice through drug repositioning. Here, we investigated the molecular details linking Thalidomide to ACE2 and COVID-19, showing that in conditions mimicking SARS-CoV-2 associated cytokine storm, the transcription factor p63 and ACE2 are stabilized and IL-8 production is increased. In such conditions, we found p63 to bind to and regulate the expression of the \u003cem\u003eACE2\u003c/em\u003egene. We previously showed that p63 is degraded upon Thalidomide treatment, and now found that treatment with this drug—or with its analogue Lenalidomide—downregulates ACE2 through p63 degradation. Finally, we found that Thalidomide treatment reduce \u003cem\u003ein vitro\u003c/em\u003e infection by pseudo-SARS-CoV-2, a baculovirus pseudotyped with the SARS-CoV-2 spike protein. Overall, we propose the dual effect of Thalidomide in reducing SARS-CoV-2 viral re-entry and inflammation through p63 degradation to weaken SARS-CoV-2 entry into host cells and mitigate lung inflammation, making it a valuable option in clinical management of COVID-19.\u003c/p\u003e","manuscriptTitle":"Molecular mechanisms of Thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-08 17:42:22","doi":"10.21203/rs.3.rs-4162662/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2024-04-25T03:25:05+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-04-03T20:19:39+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-03T18:50:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-27T12:48:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Medicine","date":"2024-03-26T13:04:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2f8333f1-c9eb-45f2-ae26-f56da783bfa6","owner":[],"postedDate":"April 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T16:02:47+00:00","versionOfRecord":{"articleIdentity":"rs-4162662","link":"https://doi.org/10.1007/s00109-024-02485-x","journal":{"identity":"journal-of-molecular-medicine","isVorOnly":false,"title":"Journal of Molecular Medicine"},"publishedOn":"2024-09-18 15:57:38","publishedOnDateReadable":"September 18th, 2024"},"versionCreatedAt":"2024-04-08 17:42:22","video":"","vorDoi":"10.1007/s00109-024-02485-x","vorDoiUrl":"https://doi.org/10.1007/s00109-024-02485-x","workflowStages":[]},"version":"v1","identity":"rs-4162662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4162662","identity":"rs-4162662","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}