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1.
Lancet Oncol ; 25(6): 707-719, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38710187

ABSTRACT

BACKGROUND: Trastuzumab deruxtecan is a HER2-directed antibody-drug conjugate approved by the US Food and Drug Administration and the European Medicines Agency for HER2-mutant non-small-cell lung cancer. Few treatment options exist for patients with HER2-mutant solid tumours beyond lung cancers. We investigated trastuzumab deruxtecan in metastatic solid tumours with specific activating HER2 mutations. METHODS: In this open-label, phase 2, basket study done in 29 centres in Asia, Europe, and North America, we investigated trastuzumab deruxtecan (5·4 mg/kg every 3 weeks by intravenous infusion) in patients aged 18 years or older with unresectable or metastatic solid tumours with specific activating HER2 mutations, an Eastern Cooperative Oncology Group performance status of 0 or 1, and disease progression following previous treatment (previous HER2-targeted therapy was permitted) or with no satisfactory alternative treatment options. The primary endpoint was confirmed objective response rate by independent central review. Anti-tumour activity and safety were analysed in all patients who received at least one dose of trastuzumab deruxtecan. This trial is registered with ClinicalTrials.gov, NCT04639219, and is active but no longer recruiting. FINDINGS: Between Dec 30, 2020, and Jan 25, 2023, 102 patients (62 [61%] female and 40 [39%] male; median age 66·5 years [IQR 58-72]; 51 [50%] White, two [2%] Black or African American, 38 [37%] Asian, and 11 [11%] did not have race information reported) with solid tumours with activating HER2 mutations received trastuzumab deruxtecan and were included in the anti-tumour activity and safety analyses sets. Patients had a median of three (IQR 2-4) previous treatment regimens. The median duration of follow-up was 8·61 months (IQR 3·71-12·68). The objective response rate by independent central review was 29·4% (95% CI 20·8-39·3; 30 of 102 patients). 52 (51%) patients had a treatment-emergent adverse event of grade 3 or worse; the most common events (in ≥5% of patients) were anaemia (16 [16%]) and neutrophil count decreased (eight [8%]). Drug-related treatment-emergent serious adverse events occurred in ten (10%) patients. Adjudicated drug-related interstitial lung disease or pneumonitis of any grade occurred in 11 patients (11%; three grade 1, five grade 2, one grade 3, and two grade 5); there were two (2%) cases of fatal adjudicated drug-related interstitial lung disease or pneumonitis. INTERPRETATION: Trastuzumab deruxtecan showed anti-tumour activity and durable responses in heavily pretreated patients across multiple tumour types with activating HER2 mutations, with no new safety signals. Prespecified HER2 mutations might be targeted by HER2-directed antibody-drug conjugates and our findings support further investigation of trastuzumab deruxtecan in the pan-tumour setting. FUNDING: AstraZeneca and Daiichi Sankyo.


Subject(s)
Immunoconjugates , Mutation , Neoplasms , Receptor, ErbB-2 , Trastuzumab , Humans , Female , Trastuzumab/therapeutic use , Trastuzumab/adverse effects , Male , Receptor, ErbB-2/genetics , Middle Aged , Aged , Immunoconjugates/therapeutic use , Immunoconjugates/adverse effects , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Camptothecin/analogs & derivatives , Camptothecin/therapeutic use , Camptothecin/adverse effects , Antineoplastic Agents, Immunological/therapeutic use , Antineoplastic Agents, Immunological/adverse effects , Adult
2.
Curr Oncol Rep ; 24(7): 809-817, 2022 07.
Article in English | MEDLINE | ID: mdl-35305211

ABSTRACT

PURPOSE OF REVIEW: Summarizing the current preclinical and clinical evidence about bystander effect of antibody-drug conjugates (ADCs) in solid tumors. RECENT FINDINGS: One of the main challenges of treating solid tumors with ADCs is the heterogeneous expression of the target antigen (Ag), which however may be overcome by the so-called bystander killing effect. This unique, but still debated, feature of certain ADCs is represented by the unintentional payload diffusion from Ag-positive tumor cells to adjacent Ag-negative tumor cells. Some pharmacological characteristics, such as a hydrophobic payload or a cleavable linker, seem to play a major role in this effect. Abundant preclinical evidence of the bystander effect has emerged, and the clinical activity of ADCs in tumors with a heterogeneous Ag expression suggests the relevance of this feature. Additional studies are required to investigate if the bystander effect is necessary for achieving a solid activity with ADCs.


Subject(s)
Antineoplastic Agents , Immunoconjugates , Neoplasms , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Bystander Effect , Humans , Immunoconjugates/therapeutic use , Neoplasms/drug therapy
3.
Chem Rev ; 118(8): 4365-4403, 2018 04 25.
Article in English | MEDLINE | ID: mdl-29600857

ABSTRACT

Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.


Subject(s)
Genomic Instability , RNA, Untranslated/genetics , DNA Breaks, Double-Stranded , DNA Damage , Gene Expression Regulation , Humans , RNA Interference , RNA-Binding Proteins/metabolism , Transcription, Genetic
4.
Nature ; 488(7410): 231-5, 2012 Aug 09.
Article in English | MEDLINE | ID: mdl-22722852

ABSTRACT

Non-coding RNAs (ncRNAs) are involved in an increasingly recognized number of cellular events. Some ncRNAs are processed by DICER and DROSHA RNases to give rise to small double-stranded RNAs involved in RNA interference (RNAi). The DNA-damage response (DDR) is a signalling pathway that originates from a DNA lesion and arrests cell proliferation3. So far, DICER and DROSHA RNA products have not been reported to control DDR activation. Here we show, in human, mouse and zebrafish, that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate the DDR upon exogenous DNA damage and oncogene-induced genotoxic stress, as studied by DDR foci formation and by checkpoint assays. DDR foci are sensitive to RNase A treatment, and DICER- and DROSHA-dependent RNA products are required to restore DDR foci in RNase-A-treated cells. Through RNA deep sequencing and the study of DDR activation at a single inducible DNA double-strand break, we demonstrate that DDR foci formation requires site-specific DICER- and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11­RAD50­NBS1-complex-dependent manner (MRE11 also known as MRE11A; NBS1 also known as NBN). DDRNAs, either chemically synthesized or in vitro generated by DICER cleavage, are sufficient to restore the DDR in RNase-A-treated cells, also in the absence of other cellular RNAs. Our results describe an unanticipated direct role of a novel class of ncRNAs in the control of DDR activation at sites of DNA damage.


Subject(s)
DNA Damage/genetics , RNA, Untranslated/genetics , Ribonuclease III/genetics , Zebrafish/genetics , Animals , Cell Cycle Proteins/metabolism , Cell Line , DNA Breaks, Double-Stranded , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , Enzyme Activation , HEK293 Cells , HeLa Cells , High-Throughput Nucleotide Sequencing , Humans , Mice , Nuclear Proteins/metabolism , RNA Interference , RNA, Untranslated/biosynthesis , Ribonuclease, Pancreatic/metabolism , Sequence Analysis, RNA , Substrate Specificity/genetics
5.
Biochim Biophys Acta ; 1860(1 Pt A): 129-39, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26518349

ABSTRACT

BACKGROUND: We have previously shown that some synthetic hydroxylated stigmastanes derived from plant sterols inhibit in vitro HSV-1 replication in ocular cell lines and decrease cytokine production in stimulated macrophages, suggesting that these steroids might combine antiviral and immunomodulating properties. In this paper we report the synthesis of some analogs fluorinated at C-6 in order to study the effect of this modification on bioactivity. METHODS: The following methods were used: organic synthesis of fluorinated analogs, cytotoxicity determination with MTT assays, cytokine production quantification with ELISAs, glucocorticoid activity determination by displacement assays, immunofluorescence and transcriptional activity assays, studies of the activation of signaling pathways by Western blot, antiviral activity evaluation through virus yield reduction assays. RESULTS: We report the chemical synthesis of new fluorinated stigmastanes and show that this family of steroidal compounds exerts its immunomodulating activity by inhibiting ERK and Akt signaling pathways, but do not act as glucocorticoids. We also demonstrate that fluorination enhances the antiviral activity. CONCLUSIONS: Fluorination on C-6 did not enhance the anti-inflammatory effect, however, an increase in the in vitro antiviral activity was observed. Thus, our results suggest that it is possible to introduce chemical modifications on the parent steroids in order to selectively modulate one of the effects. GENERAL SIGNIFICANCE: This family of steroids could allow the development of an alternative treatment for ocular immunopathologies triggered by HSV-1, without the undesirable side effects of the currently used drugs.


Subject(s)
Antiviral Agents/pharmacology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Herpesvirus 1, Human/drug effects , Immunologic Factors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Receptors, Glucocorticoid/metabolism , Signal Transduction/drug effects , Sitosterols/pharmacology , Stigmasterol/pharmacology , Animals , Chlorocebus aethiops , Cytokines/biosynthesis , HEK293 Cells , Humans , Macrophages/drug effects , Macrophages/immunology , Proto-Oncogene Proteins c-akt/physiology , Vero Cells
6.
Med Microbiol Immunol ; 204(5): 575-84, 2015 Oct.
Article in English | MEDLINE | ID: mdl-25528328

ABSTRACT

The pathogenesis of many viral infections lies on the damage caused by the immune response against the virus. Current antiviral drugs do not act on the inflammatory component of the disease. Thus, new compounds that inhibit both viral multiplication and the immunopathology elicited by the virus are an approach that should be considered. In the present study, we identified two jatropholones (2A and 5B) and one carnosic acid derivative (9C) that significantly inhibited multiplication of TK+ and TK- strains of HSV-1 in Vero cells. Compounds 2A, 5B and 9C also prevented HSV-1- and TLRs-induced inflammatory response in cultivated murine macrophages. In macrophages infected with HSV-1, the inhibitory effect of compounds 2A, 5B and 9C on TNF-α and IL-6 production could be associated with the block of ERK pathway, whereas NF-κB pathway was not hampered by any of the compounds. Besides, 2A, 5B and 9C also inhibited ERK pathway and reduced TNF-α production in macrophages stimulated with TLR2, TLR4 or TLR9 agonists and were able to hinder IL-6 secretion after activation with TLR2 or TLR4, but not with TLR9. The immunomodulatory effect of 2A, 5B and 9C in macrophages infected with HSV-1 may be a consequence of the inhibition of ERK pathway activated by TLRs. The availability of compounds with both antiviral and immunomodulatory properties which affect TLR signaling pathways might be a useful strategy to control the progress of virus-induced disease.


Subject(s)
Antiviral Agents/pharmacology , Cytokines/antagonists & inhibitors , Diterpenes/pharmacology , Herpesvirus 1, Human/immunology , Immunosuppressive Agents/pharmacology , MAP Kinase Signaling System/drug effects , Virus Replication , Animals , Cell Line , Chlorocebus aethiops , Herpesvirus 1, Human/physiology , Mice
7.
Foot Ankle Int ; 45(8): 905-915, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38676564

ABSTRACT

BACKGROUND: Diabetic foot ulcers (DFUs) constitute a complication that occurs in 19% to 34% of patients with diabetes mellitus (DM). The aim of this study is to describe median days to healing, average velocity of wound closure, and percentage of wound surface closed at 3, 6, and 12 weeks through the use of homogenized and lyophilized amniotic membrane (hAMpe) dressings for the treatment of DFUs in ambulatory patients. METHODS: An observational, descriptive, longitudinal study was performed. Patients presenting with granulation-based DFU, after proper debridement, were included from August 19, 2021, until July 14, 2023. hAMpe dressings placed every 3 days were used for the treatment of these ulcers. RESULTS: Sixteen patients were included with a mean age of 52.38 (8.07) years. The analyzed lesions were postsurgical ulcers in 15 of the 16 included patients. Median ulcer size was 19.5 cm2 (6.12-36). The median ABI was 1.10 (1-1.14). The median days to healing was 96 (71-170). The median percentage closure of the wound at 3 weeks was 41% (28.9%-55.3%), at 6 weeks it was 68.2% (48.6%-74.2%), and at 12 weeks it was 100% (81%-100%). The average velocity closure was 1.04% per day (95% CI 0.71%-1.31%). It was higher during the closure of the first 50% of the ulcer, 2.12% per day (95% CI 0.16%-4.09%), and decreased from 50% to 25% of the ulcer size to 0.67% per day (95% CI 0.23%-1.10%) and from 25% to closure to 0.47% per day (95% CI 0.14%-0.80%), P < .001. CONCLUSION: These results are difficult to compare to other studies given the higher surface area of the ulcers included in our sample. The development of hAMpe dressings enables patients to apply them without requiring assistance from health care teams and was not associated with any recognized complications.


Subject(s)
Amnion , Diabetic Foot , Wound Healing , Humans , Diabetic Foot/therapy , Middle Aged , Female , Male , Amnion/transplantation , Longitudinal Studies , Biological Dressings , Adult , Freeze Drying , Bandages , Aged
8.
Cell Rep Med ; : 101792, 2024 Oct 11.
Article in English | MEDLINE | ID: mdl-39437778

ABSTRACT

Trastuzumab deruxtecan (T-DXd) is a human epidermal growth factor receptor 2 (HER2)-targeting antibody drug conjugate that has remarkable activity in HER2-positive cancers. However, the degree of benefit of T-DXd is not uniform among solid tumors even with high levels of HER2. Despite high HER2 expression, the HER2/T-DXd complex may not always undergo internalization and payload release dependent on the receptor's conformation and context. We hypothesize that epidermal growth factor receptor (EGFR), a dimerization partner of HER2, can modulate HER2 trafficking through endocytic pathways and affect T-DXd uptake. We demonstrate that elevated EGFR expression levels can promote EGFR/HER2 heterodimer formation and suppress T-DXd internalization and efficacy. Knockdown of EGFR expression or pharmacologic stimulation of EGFR endocytosis with EGFR monoclonal antibodies restores T-DXd trafficking and antitumor activity in EGFR-overexpressing cancers in vivo. Our results reveal EGFR overexpression to be a potential mechanism of resistance to T-DXd, which can be overcome by combination therapy strategies targeting EGFR.

9.
Bioorg Med Chem ; 21(2): 560-8, 2013 Jan 15.
Article in English | MEDLINE | ID: mdl-23219855

ABSTRACT

Many viral infections are associated with the development of immunopathologies and autoimmune diseases, which are of difficult treatment and for which no vaccines are yet available. Obtaining compounds that conjugate both antiviral and immunomodulatory activities in the same molecule would be very useful for the prevention and/or treatment of these immunopathologies. The compound (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) displays anti-Herpes simplex virus type 1 activity in vitro and reduces the incidence of herpetic stromal keratitis (HSK) in mice, a chronic inflammatory syndrome induced by ocular HSV-1 infection. In the present study, compound 1 showed opposite immunomodulatory properties in vitro. It induced the release of pro-inflammatory cytokines in HSV-1-infected epithelial cells of ocular origin, and significantly reduced the production of these cytokines in LPS-activated macrophages. RNA microarrays revealed various overexpressed and repressed genes in compound 1 treated infected epithelial cells and activated macrophages, many of which are associated with innate immune responses and inflammatory processes. These immunomodulatory properties of compound 1, together with its previously reported antiviral activity, make it a potential drug for the treatment of HSK and many other immunopathologies of viral and non-viral origin.


Subject(s)
Antiviral Agents/pharmacology , Cholestenones/chemistry , Herpesvirus 1, Human/drug effects , Immunologic Factors/chemistry , Stigmasterol/analogs & derivatives , Animals , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Cell Line , Cell Survival/drug effects , Cholestenones/pharmacology , Cholestenones/therapeutic use , Corneal Stroma/cytology , Corneal Stroma/virology , Cytokines/genetics , Cytokines/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/virology , Gene Expression Profiling , Humans , Immunologic Factors/pharmacology , Immunologic Factors/therapeutic use , Keratitis, Herpetic/drug therapy , Keratitis, Herpetic/immunology , Keratitis, Herpetic/veterinary , Mice , NF-kappa B/genetics , NF-kappa B/metabolism , Stigmasterol/chemistry , Stigmasterol/pharmacology , Stigmasterol/therapeutic use , Transcriptional Activation/drug effects
10.
Heliyon ; 9(10): e20148, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37822633

ABSTRACT

HRSV is responsible for many acute lower airway infections and hospitalizations in infants, the elderly and those with weakened immune systems around the world. The strong inflammatory response that mediates viral clearance contributes to pathogenesis, and is positively correlated with disease severity. There is no specific effective therapy on hand. Antiviral synthetic stigmastanes (22S, 23S)-22,23-dihydroxystigmast-4-en-3-one (Compound 1) and 22,23-dihydroxystigmasta-1,4-dien-3-one (Compound 2) have shown to be active inhibiting unrelated virus like Herpes Simplex type 1 virus (HSV-1) and Adenovirus, without cytotoxicity. We have also shown that Compound 1 modulates the activation of cell signaling pathways and cytokine secretion in infected epithelial cells as well as in inflammatory cells activated by nonviral stimuli. In the present work, we investigated the inhibitory effect of both compounds on HRSV replication and their modulatory effect on infected epithelial and inflammatory cells. We show that compounds 1 and 2 inhibit in vitro HRSV replication and propagation and reduce cytokine secretion triggered by HRSV infection in epithelial and inflammatory cells. The compounds reduce viral loads and inflammatory infiltration in the lungs of mice infected with HRSV.

11.
Cancer Res ; 82(12): 2269-2280, 2022 06 15.
Article in English | MEDLINE | ID: mdl-35442400

ABSTRACT

The phosphoinositide 3-kinase (PI3K) pathway regulates proliferation, survival, and metabolism and is frequently activated across human cancers. A comprehensive elucidation of how this signaling pathway controls transcriptional and cotranscriptional processes could provide new insights into the key functions of PI3K signaling in cancer. Here, we undertook a transcriptomic approach to investigate genome-wide gene expression and transcription factor activity changes, as well as splicing and isoform usage dynamics, downstream of PI3K. These analyses uncovered widespread alternatively spliced isoforms linked to proliferation, metabolism, and splicing in PIK3CA-mutant cells, which were reversed by inhibition of PI3Kα. Analysis of paired tumor biopsies from patients with PIK3CA-mutated breast cancer undergoing treatment with PI3Kα inhibitors identified widespread splicing alterations that affect specific isoforms in common with the preclinical models, and these alterations, namely PTK2/FRNK and AFMID isoforms, were validated as functional drivers of cancer cell growth or migration. Mechanistically, isoform-specific splicing factors mediated PI3K-dependent RNA splicing. Treatment with splicing inhibitors rendered breast cancer cells more sensitive to the PI3Kα inhibitor alpelisib, resulting in greater growth inhibition than alpelisib alone. This study provides the first comprehensive analysis of widespread splicing alterations driven by oncogenic PI3K in breast cancer. The atlas of PI3K-mediated splicing programs establishes a key role for the PI3K pathway in regulating splicing, opening new avenues for exploiting PI3K signaling as a therapeutic vulnerability in breast cancer. SIGNIFICANCE: Transcriptomic analysis reveals a key role for the PI3K pathway in regulating RNA splicing, uncovering new mechanisms by which PI3K regulates proliferation and metabolism in breast cancer. See related commentary by Claridge and Hopkins, p. 2216.


Subject(s)
Breast Neoplasms , Phosphatidylinositol 3-Kinases , Breast Neoplasms/pathology , Carcinogenesis/genetics , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases/genetics , Female , Humans , Phosphatidylinositol 3-Kinase/genetics , Phosphatidylinositol 3-Kinase/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , RNA Splicing/genetics , Transcriptome
12.
Cell Rep ; 34(1): 108565, 2021 01 05.
Article in English | MEDLINE | ID: mdl-33406426

ABSTRACT

The MRE11-RAD50-NBS1 (MRN) complex supports the synthesis of damage-induced long non-coding RNA (dilncRNA) by RNA polymerase II (RNAPII) from DNA double-strand breaks (DSBs) by an unknown mechanism. Here, we show that recombinant human MRN and native RNAPII are sufficient to reconstitute a minimal functional transcriptional apparatus at DSBs. MRN recruits and stabilizes RNAPII at DSBs. Unexpectedly, transcription is promoted independently from MRN nuclease activities. Rather, transcription depends on the ability of MRN to melt DNA ends, as shown by the use of MRN mutants and specific allosteric inhibitors. Single-molecule FRET assays with wild-type and mutant MRN show a tight correlation between the ability to melt DNA ends and to promote transcription. The addition of RPA enhances MRN-mediated transcription, and unpaired DNA ends allow MRN-independent transcription by RNAPII. These results support a model in which MRN generates single-strand DNA ends that favor the initiation of transcription by RNAPII.


Subject(s)
Acid Anhydride Hydrolases/metabolism , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , MRE11 Homologue Protein/metabolism , Nuclear Proteins/metabolism , Nucleic Acid Denaturation , RNA Polymerase II/metabolism , RNA, Long Noncoding/biosynthesis , Transcription, Genetic , Acid Anhydride Hydrolases/genetics , Cell Cycle Proteins/genetics , DNA Breaks, Double-Stranded , DNA Damage , DNA Repair , DNA-Binding Proteins/genetics , HeLa Cells , Humans , MRE11 Homologue Protein/genetics , Mutation , Nuclear Proteins/genetics , RNA Polymerase II/genetics , RNA, Long Noncoding/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
13.
Cancer Res ; 81(9): 2470-2480, 2021 05 01.
Article in English | MEDLINE | ID: mdl-33685991

ABSTRACT

PI3Kα inhibitors have shown clinical activity in PIK3CA-mutated estrogen receptor-positive (ER+) patients with breast cancer. Using whole genome CRISPR/Cas9 sgRNA knockout screens, we identified and validated several negative regulators of mTORC1 whose loss confers resistance to PI3Kα inhibition. Among the top candidates were TSC1, TSC2, TBC1D7, AKT1S1, STK11, MARK2, PDE7A, DEPDC5, NPRL2, NPRL3, C12orf66, SZT2, and ITFG2. Loss of these genes invariably results in sustained mTOR signaling under pharmacologic inhibition of the PI3K-AKT pathway. Moreover, resistance could be prevented or overcome by mTOR inhibition, confirming the causative role of sustained mTOR activity in limiting the sensitivity to PI3Kα inhibition. Cumulatively, genomic alterations affecting these genes are identified in about 15% of PIK3CA-mutated breast tumors and appear to be mutually exclusive. This study improves our understanding of the role of mTOR signaling restoration in leading to resistance to PI3Kα inhibition and proposes therapeutic strategies to prevent or revert this resistance. SIGNIFICANCE: These findings show that genetic lesions of multiple negative regulators of mTORC1 could limit the efficacy of PI3Kα inhibitors in breast cancer, which may guide patient selection strategies for future clinical trials.


Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Class I Phosphatidylinositol 3-Kinases/genetics , Drug Resistance, Neoplasm/drug effects , Loss of Function Mutation , Mechanistic Target of Rapamycin Complex 1/metabolism , Phosphoinositide-3 Kinase Inhibitors/administration & dosage , Signal Transduction/genetics , Thiazoles/administration & dosage , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Survival/drug effects , Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Cohort Studies , Female , HEK293 Cells , Humans , MCF-7 Cells , Mice , TOR Serine-Threonine Kinases/metabolism , Transduction, Genetic , Tumor Burden/drug effects , Tumor Burden/genetics , Xenograft Model Antitumor Assays
14.
Antiviral Res ; 183: 104879, 2020 11.
Article in English | MEDLINE | ID: mdl-32918925

ABSTRACT

Although human adenovirus (ADV) infections are mild and self-limited in immunocompetent individuals, they can be severe and life-threatening in immunocompromised patients. Despite their significant clinical impact, there are not currently approved antiviral therapies for ADV infections. On the other hand, in some cases, the immune response induced by ADV infection can cause tissue damage. Even more, in the case of adenovirus vectors used in gene therapy, host immunity generally antagonize viral efficacy. Therefore, the need for searching an effective and safe therapy is increasing. In this work, we describe the antiadenoviral activity of the synthetic stigmastane (22S, 23S)-22,23-dihydroxystigmast-4-en-3-one (Compound 1) with already reported antiviral and antiinflammatory activities against other viruses of clinical importance. Compound 1 displayed no virucidal activity and did not affect ADV entry to the cells. The compound inhibited viral replication and it also reduced cytokine secretion in epithelial and inflammatory infected cells. Thus, Compound 1 would be a promissory drug potentially useful against adenoviral infections as well as an adjuvant of adenoviral vectors in gene therapy.


Subject(s)
Adenoviridae/drug effects , Anti-Inflammatory Agents/pharmacology , Antiviral Agents/pharmacology , Virus Replication/drug effects , A549 Cells , Adenoviridae/immunology , Adenovirus Infections, Human/drug therapy , Anti-Inflammatory Agents/chemical synthesis , Antiviral Agents/chemical synthesis , Cell Line , Cells, Cultured , Cornea/cytology , Cornea/virology , Epithelial Cells/drug effects , Epithelial Cells/virology , Genetic Vectors , Humans , Inflammation/drug therapy
15.
Cancer Discov ; 10(5): 674-687, 2020 05.
Article in English | MEDLINE | ID: mdl-32213539

ABSTRACT

Amplification of and oncogenic mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody-drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy. SIGNIFICANCE: T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2. This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy.See related commentary by Rolfo and Russo, p. 643.This article is highlighted in the In This Issue feature, p. 627.


Subject(s)
Lung Neoplasms/drug therapy , Receptor, ErbB-2/metabolism , Adult , Aged , Aged, 80 and over , Cell Line, Tumor , Female , Humans , Lung Neoplasms/pathology , Male , Middle Aged , Mutation
16.
Nat Protoc ; 14(5): 1489-1508, 2019 05.
Article in English | MEDLINE | ID: mdl-30962605

ABSTRACT

Non-coding RNA (ncRNA) molecules have been shown to play a variety of cellular roles; however, the contributions of different types of RNA to specific phenomena are often hard to dissect. To study the role of RNA in the assembly of DNA damage response (DDR) foci, we developed the RNase A treatment and reconstitution (RATaR) method, in which cells are mildly permeabilized, incubated with recombinant RNase A and subsequently reconstituted with different RNA species, under conditions of RNase A inactivation and inhibition of endogenous transcription. The block of transcription right after RNase A removal represents a key innovation of RATaR, preventing potential contributions of endogenously neo-synthesized transcripts to the phenotypes studied. A critical aspect of this technique is the balance between sufficient permeabilization of membranes to allow enzyme/RNA access into the cell nucleus and cell viability. Here, we present our protocol for RNA-dependent DDR foci disassembly and reassembly using fluorescent DDR RNAs (DDRNAs) in NIH2/4 cells, an engineered NIH3T3-derived cell line. The use of sequence-specific, fluorescent RNA molecules permits the concomitant determination of their subcellular localization and biological functions. We also outline adaptations of RATaR when implemented in different cell lines exposed to various genotoxic treatments, such as γ-radiation, restriction enzymes and telomere deprotection. In all these cases, the entire procedure can be completed within 2 h without the need for special equipment or uncommon skills. We believe this technique will prove useful for investigating the contribution of RNA to a variety of relevant cellular processes.


Subject(s)
DNA Damage , DNA Repair , RNA, Untranslated , Ribonuclease, Pancreatic/metabolism , Animals , DNA Damage/genetics , DNA Damage/physiology , DNA Repair/genetics , DNA Repair/physiology , Genetic Techniques , HeLa Cells , Humans , Mice , NIH 3T3 Cells , RNA/analysis , RNA/genetics , RNA/metabolism , RNA, Untranslated/genetics , RNA, Untranslated/physiology
17.
Sci Rep ; 9(1): 6460, 2019 04 23.
Article in English | MEDLINE | ID: mdl-31015566

ABSTRACT

A novel class of small non-coding RNAs called DNA damage response RNAs (DDRNAs) generated at DNA double-strand breaks (DSBs) in a DROSHA- and DICER-dependent manner has been shown to regulate the DNA damage response (DDR). Similar molecules were also reported to guide DNA repair. Here, we show that DDR activation and DNA repair can be pharmacologically boosted by acting on such non-coding RNAs. Cells treated with enoxacin, a compound previously demonstrated to augment DICER activity, show stronger DDR signalling and faster DNA repair upon exposure to ionizing radiations compared to vehicle-only treated cells. Enoxacin stimulates DDRNA production at chromosomal DSBs and at dysfunctional telomeres, which in turn promotes 53BP1 accumulation at damaged sites, therefore in a miRNA-independent manner. Increased 53BP1 occupancy at DNA lesions induced by enoxacin ultimately suppresses homologous recombination, channelling DNA repair towards faster and more accurate non-homologous end-joining, including in post-mitotic primary neurons. Notably, augmented DNA repair stimulated by enoxacin increases the survival also of cancer cells treated with chemotherapeutic agents.


Subject(s)
DNA Damage , DNA End-Joining Repair/drug effects , Enoxacin/pharmacology , MicroRNAs/metabolism , Signal Transduction/drug effects , HeLa Cells , Humans , MicroRNAs/genetics , Telomere/genetics , Telomere/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Tumor Suppressor p53-Binding Protein 1/metabolism
18.
Antiviral Res ; 164: 1-11, 2019 04.
Article in English | MEDLINE | ID: mdl-30711418

ABSTRACT

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease and bronchiolitis in children worldwide. No vaccine or specific, effective treatment is currently available. ß-escin is one of the main bioactive constituents of Aesculus hippocastanum L. (Hippocastanaceae) seed extract (AH), and both ß-escin and AH have demonstrated a beneficial role in clinical therapy because of their anti-edematous, anti-inflammatory and antioxidative effects. Besides, we have reported that ß-escin and AH show virucidal, antiviral and immunomodulatory activities against the enveloped viruses HSV-1, VSV and Dengue virus in vitro. In this study, we demonstrate that ß-escin and AH have virucidal and antiviral activities against RSV, as well as NF-κB, AP-1 and cytokine modulating activities in RSV infected epithelial and macrophage cell lines in vitro. Besides, in a murine model of pulmonary RSV infection, AH treatment improves the course of acute disease, evidenced by decreased weight loss, reduced RSV lung titers, and attenuated airway inflammation. In contrast, even though ß-escin showed, similarly to AH, antiviral and immunomodulatory properties in vitro, it neither reduces viral titers nor attenuates lung injury in vivo. Thus, our data demonstrate that AH restrains RSV disease through antiviral and immunomodulatory effect.


Subject(s)
Aesculus/chemistry , Antiviral Agents/therapeutic use , Plant Extracts/pharmacology , Pneumonia/drug therapy , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Virus, Human/drug effects , Animals , Cell Line , Female , Humans , Immunomodulation , Lung/drug effects , Lung/pathology , Lung/virology , Mice , Mice, Inbred BALB C , Plants, Medicinal/chemistry , Pneumonia/virology , Seeds/chemistry
19.
J Steroid Biochem Mol Biol ; 108(1-2): 164-70, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18054220

ABSTRACT

Herpes simplex virus type 1 (HSV-1) induces an ocular chronic immunoinflammatory syndrome named herpetic stromal keratitis that can lead to vision impairment and blindness. We have reported that the synthetic brassinosteroid (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one, designated as 2, is a potent antiviral in vitro and reduces the incidence of murine herpetic stromal keratitis, although it does not exert an antiviral effect in vivo. In the present report, we investigated whether brassinosteroid 2 may play a role in the modulation of the response of epithelial and immune cells to HSV-1 infection. Compound 2 blocked HSV-1-induced activation of NF-kappaB by inhibiting its translocation to the nucleus of infected corneal and conjunctival cells in vitro, as well as significantly reduced the secretion of TNF-alpha in infected NHC cells. Conversely, IL-6 production was enhanced by compound 2 after HSV-1 infection in both cell types. The production of these cytokines was considerably reduced in a LPS-stimulated macrophage cell line after treatment with compound 2. In conclusion, brassinosteroid 2 would be playing a modulating effect as an inductor or inhibitor, depending on the cell type involved. The improvement of disease observed in mice could be a balance between both, the immunostimulating and immunosuppressive effects of brassinosteroid 2 in vivo.


Subject(s)
Cholestanones/pharmacology , Cholestanones/therapeutic use , Keratitis, Herpetic/drug therapy , Steroids/pharmacology , Steroids/therapeutic use , Animals , Antiviral Agents/pharmacology , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cells, Cultured , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelium, Corneal/drug effects , Epithelium, Corneal/metabolism , Gene Expression Regulation, Viral/drug effects , Herpesvirus 1, Human/genetics , Humans , Immunologic Factors/pharmacology , Interleukin-6/metabolism , Macrophages/drug effects , Macrophages/metabolism , Mice , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/metabolism , Viral Envelope Proteins/genetics
20.
J Steroid Biochem Mol Biol ; 111(1-2): 111-6, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18619833

ABSTRACT

Stromal keratitis resulting from ocular infection with Herpes simplex virus type 1 (HSV-1) is a common cause of blindness. This report investigates the antiviral and anti-inflammatory properties of two new synthetic stigmastane analogs in the experimental model of HSV-1-induced ocular disease in mice. (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (1) and (22S,23S)-22,23-dihydroxystigmasta-1,4-dien-3-one (2) exhibited anti-HSV-1 activity in vitro and ameliorated the signs of murine herpetic stromal keratitis (HSK), although none of the compounds showed antiviral activity in vivo. We discuss that the improvement of HSK could be due to an immunomodulatory effect of both compounds.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Stigmasterol/analogs & derivatives , Stigmasterol/chemical synthesis , Animals , Antiviral Agents/chemistry , Cell Line , Cell Survival/drug effects , Chlorocebus aethiops , Disease Models, Animal , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelium, Corneal/drug effects , Epithelium, Corneal/metabolism , Formazans/metabolism , Herpesvirus 1, Human/metabolism , Humans , Inhibitory Concentration 50 , Keratitis, Herpetic/drug therapy , Male , Mice , Mice, Inbred BALB C , Molecular Structure , Spectrophotometry , Tetrazolium Salts/metabolism , Time Factors , Vero Cells , beta-Galactosidase/metabolism
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