Active notch protects MAPK activated melanoma cell lines from MEK inhibitor cobimetinib

The crosstalk between Notch and MAPK pathway plays a role in MEK inhibitor resistance in BRAFV600E metastatic melanoma (MM) and promotes migration in GNAQQ209L uveal melanoma (UM) cells. We determined the cytotoxicity of combinatorial inhibition of MEK and Notch by cobimetinib and γ-secretase inhibitor (GSI) nirogacestat, in BRAFV600E and BRAF wt MM and GNAQQ209L UM cells displaying different Erk1/2 and Notch activation status, with the aim to elucidate the impact of Notch signaling in the response to MEK inhibitor. Overall the combination was synergic in BRAFV600E MM and GNAQQ209L UM cells and antagonistic in BRAF wt one. Focusing on UM cells, we found that cobimetinib resulted in G0/G1 phase arrest and apoptosis induction, whereas the combination with GSI increased treatment efficacy by inducing a senescent-like state of cells and by blocking migration towards liver cancer cells. Mechanistically, this was reflected in a strong reduction of cyclin D1, in the inactivation of retinoblastoma protein and in the increase of p27KIP1 expression levels. Of note, each drug alone prevented Notch signaling activation resulting in inhibition of c-jun(Ser63) and Hes-1 expression. The combination achieved the strongest inhibition on Notch signaling and on both c-jun(Ser63) and Erk1/2 activation level. In conclusion we unveiled a coordinate action of MAPK and Notch signaling in promoting proliferation of BRAFV600E MM and GNAQQ209L UM cells. Remarkably, the simultaneous inhibition of MEK and Notch signaling highlighted a role for the second pathway in protecting cells against senescence in GNAQQ209L UM cells treated with the MEK inhibitor

Epilepsy in elderly patients: focus on treatment

Epilepsy is a disorder widely distributed across all age groups, although its incidence and prevalence favours elderly patients. In this population the development of epilepsy is mainlyrelated to other brain disorders that facilitate the occurrence of late seizures, mostly of focal onset. Apart from the difficulties faced in current practice with the appropriate diagnosis of the epileptic nature of the episodes, old subjects require an individualised drug treatment, tailored on their characteristics, such as comorbidity, polytherapy, age-associated biological impairment and compliance. Among the various antiepileptic drugs available for the treatment of epilepsy, rational selection and combination of drugs should drive both initiation and maintenance of the treatment. Choice of antiepileptic drugs should first focus on avoidance of adverse effects and potential drug interactions. The pharmacological control of seizures is usually favourable at this age, with a good prognosis. However, alternative therapeutic options should be considered as soon as drug-response is unsatisfactory

Functional analysis of splicing mutations in exon 7 of NF1 gene

BACKGROUND: Neurofibromatosis type 1 is one of the most common autosomal dominant disorders, affecting about 1:3,500 individuals. NF1 exon 7 displays weakly defined exon-intron boundaries, and is particularly prone to missplicing. METHODS: In this study we investigated the expression of exon 7 transcripts using bioinformatic identification of splicing regulatory sequences, and functional minigene analysis of four sequence changes [c.910C>T (R304X), c.945G>A/c.946C>A (Q315Q/L316M), c.1005T>C (N335N)] identified in exon 7 of three different NF1 patients. RESULTS: Our results detected the presence of three exonic splicing enhancers (ESEs) and one putative exonic splicing silencer (ESS) element. The wild type minigene assay resulted in three alternative isoforms, including a transcript lacking NF1 exon 7 (NF1ΔE7). Both the wild type and the mutated constructs shared NF1ΔE7 in addition to the complete messenger, but displayed a different ratio between the two transcripts. In the presence of R304X and Q315Q/L316M mutations, the relative proportion between the different isoforms is shifted toward the expression of NF1ΔE7, while in the presence of N335N variant, the NF1ΔE7 expression is abolished. CONCLUSION: In conclusion, it appears mandatory to investigate the role of each nucleotide change within the NF1 coding sequence, since a significant proportion of NF1 exon 7 mutations affects pre-mRNA splicing, by disrupting exonic splicing motifs and modifying the delicate balance between aberrantly and correctly spliced transcripts

Anti-Biofilm Activity of Phenyllactic Acid against Clinical Isolates of Fluconazole-Resistant Candida albicans

: Commonly found colonizing the human microbiota, Candida albicans is a microorganism known for its ability to cause infections, mainly in the vulvovaginal region, and is responsible for 85% to 90% of vulvovaginal candidiasis (VVC) cases. The development of drug resistance in C. albicans isolates after long-term therapy with fluconazole is an important complication to solve and new therapeutic strategies are required to target this organism and its pathogenicity. In the present study, phenyllactic acid (PLA) an important broad-spectrum antimicrobial compound was investigated for its antifungal and antivirulence activities against clinical isolates of C. albicans. Previously characterized strains of C. albicans isolates from women with VVC and C. albicans ATCC90028 were used to evaluate the antimicrobial and time dependent killing assay activity of PLA showing a MIC 7.5 mg mL-1 and a complete reduction of viable Candida cells detected by killing kinetics after 4 h of treatment with PLA. Additionally, PLA significantly reduced the biomass and the metabolic activity of C. albicans biofilms and impaired biofilm formation also with changes in ERG11, ALS3, and HWP1 genes expression as detected by qPCR. PLA eradicated pre-formed biofilms as showed also with confocal laser scanning microscopy (CLSM) observations. Furthermore, the compound prolonged the survival rate of Galleria mellonella infected by C. albicans isolates. These results indicate that PLA is a promising candidate as novel and safe antifungal agents for the treatment of vulvovaginal candidiasis

Quality of life and anxious-depressive symptoms in cancer patients undergoing mindfulness-based interventions: Feasibility and preliminary outcomes on prospective single-centre case-control study (MIND4ME St.)

Background Mindfulness based interventions (MBIs) have shown efficacy in improving psychological symptoms including depression and anxiety in cancer patients (pts). The study aimed to explore feasibility and reproducibility of MBIs in an Italian Cancer Centre measuring biochemical and psychological parameters. Methods In this pilot prospective case-control study, we recruited newly diagnosed pts receiving adjuvant chemotherapy (CT). A MBIs program was designed consisting of 2.5 hours weekly for 8 weeks and, including meditation, yoga and body scan. Material for 45 minutes (mn) home daily practice was provided. Primary endpoint was to evaluate feasibility. Secondary endpoints were assessment of quality of life (QoL), psychological and biochemical outcomes of stress, tested at baseline (W0), W4, W8, W24, W48. PSS (Perceived Stress Reduction), POMS (profile of mood states scores), EuroQoL (EQ-5D-3L) were administered. Results Ten pts underwent MBIs program arm. We present preliminary results, while data of control arm are being collected. All pts were female, two pts (20%) dropped out. Median age was 56 years. All received adjuvant CT, 5/8 received radiotherapy and hormone therapy. Mean of sessions attending was 6.8 (76%). Median daily practice was 30 mn. EQ-5D item for depression and anxiety showed decreasing trend in mean score from moderate to light (P = 0.15) and significant improvement of auto-perceived QoL was observed comparing W0 and W8 (P = 0.02) Conclusions In a sensitive setting such as start CT, we found high pts compliance to MBIs. Improvement in self-perceived QoL after starting program was found and comparing anxious-depressive symptoms outcomes with control arm is still needed

LAMC2 marks a tumor-initiating cell population with an aggressive signature in pancreatic cancer

[Background]: Tumor-initiating cells (TIC), also known as cancer stem cells, are considered a specific subpopulation of cells necessary for cancer initiation and metastasis; however, the mechanisms by which they acquire metastatic traits are not well understood.[Methods]: LAMC2 transcriptional levels were evaluated using publicly available transcriptome data sets, and LAMC2 immunohistochemistry was performed using a tissue microarray composed of PDAC and normal pancreas tissues. Silencing and tracing of LAMC2 was performed using lentiviral shRNA constructs and CRISPR/Cas9-mediated homologous recombination, respectively. The contribution of LAMC2 to PDAC tumorigenicity was explored in vitro by tumor cell invasion, migration, sphere-forming and organoids assays, and in vivo by tumor growth and metastatic assays. mRNA sequencing was performed to identify key cellular pathways upregulated in LAMC2 expressing cells. Metastatic spreading induced by LAMC2- expressing cells was blocked by pharmacological inhibition of transforming growth factor beta (TGF-β) signaling.[Results]: We report a LAMC2-expressing cell population, which is endowed with enhanced self-renewal capacity, and is sufficient for tumor initiation and differentiation, and drives metastasis. mRNA profiling of these cells indicates a prominent squamous signature, and differentially activated pathways critical for tumor growth and metastasis, including deregulation of the TGF-β signaling pathway. Treatment with Vactosertib, a new small molecule inhibitor of the TGF-β type I receptor (activin receptor-like kinase-5, ALK5), completely abrogated lung metastasis, primarily originating from LAMC2-expressing cells.[Conclusions]: We have identified a highly metastatic subpopulation of TICs marked by LAMC2. Strategies aimed at targeting the LAMC2 population may be effective in reducing tumor aggressiveness in PDAC patients. Our results prompt further study of this TIC population in pancreatic cancer and exploration as a potential therapeutic target and/or biomarker.This work was supported by: Marie Curie IF (H2020-MSCA-IF-2015, #703753), My First AIRC Grant (MFAG-2017, #20206), POR Campania FESR 2014/2020 (Project SATIN) to E.L.; AIRC IG grant 2018 n.21420 to A.D.L.; FIMP to D.D.C.; AECC (Proye18046BATL_002) to E.B.; My First AIRC Grant (MFAG grant #23029), WorldWide Cancer Research (Research grant #20–0188), EASI Genomics consortium (TNA project #15158) and the World Cancer Research Fund (Seed grant #2021–1769) to A.C

Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase

Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the ‘DFG-out’ inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the ‘gatekeeper’ V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET

Impact of social determinants on antiretroviral therapy access and outcomes entering the era of universal treatment for people living with HIV in Italy

Background: Social determinants are known to be a driving force of health inequalities, even in high income countries. Aim of our study was to determine if these factors can limit antiretroviral therapy (ART) access, outcome and retention in care of people living with HIV (PLHIV) in Italy. Methods: All ART naïve HIV+ patients (pts) of Italian nationality enrolled in the ICONA Cohort from 2002 to 2016 were included. The association of socio-demographic characteristics (age, sex, risk factor for HIV infection, educational level, occupational status and residency area) with time to: ART initiation (from the first positive anti-HIV test), ART regimen discontinuation, and first HIV-RNA < 50 cp/mL, were evaluated by Cox regression analysis, Kaplan Meier method and log-rank test. Results: A total of 8023 HIV+ pts (82% males, median age at first pos anti-HIV test 36 years, IQR: 29-44) were included: 6214 (77.5%) started ART during the study period. Women, people who inject drugs (PWID) and residents in Southern Italy presented the lowest levels of education and the highest rate of unemployment compared to other groups. Females, pts aged > 50 yrs., unemployed vs employed, and people with lower educational levels presented the lowest CD4 count at ART initiation compared to other groups. The overall median time to ART initiation was 0.6 years (yrs) (IQR 0.1-3.7), with a significant decrease over time [2002-2006 = 3.3 yrs. (0.2-9.4); 2007-2011 = 1.0 yrs. (0.1-3.9); 2012-2016 = 0.2 yrs. (0.1-2.1), p < 0.001]. By multivariate analysis, females (p < 0.01) and PWID (p < 0.001), presented a longer time to ART initiation, while older people (p < 0.001), people with higher educational levels (p < 0.001), unemployed (p = 0.02) and students (p < 0.001) were more likely to initiate ART. Moreover, PWID, unemployed vs stable employed, and pts. with lower educational levels showed a lower 1-year probability of achieving HIV-RNA suppression, while females, older patients, men who have sex with men (MSM), unemployed had higher 1-year risk of first-line ART discontinuation. Conclusions: Despite median time to ART start decreased from 2002 to 2016, socio-demographic factors still contribute to disparities in ART initiation, outcome and durability

High familial burden of cancer correlates with improved outcome from immunotherapy in patients with NSCLC independent of somatic DNA damage response gene status

Family history of cancer (FHC) is a hallmark of cancer risk and an independent predictor of outcome, albeit with uncertain biologic foundations. We previously showed that FHC-high patients experienced prolonged overall (OS) and progression-free survival (PFS) following PD-1/PD-L1 checkpoint inhibitors. To validate our findings in patients with NSCLC, we evaluated two multicenter cohorts of patients with metastatic NSCLC receiving either first-line pembrolizumab or chemotherapy. From each cohort, 607 patients were randomly case-control matched accounting for FHC, age, performance status, and disease burden. Compared to FHC-low/negative, FHC-high patients experienced longer OS (HR 0.67 [95% CI 0.46-0.95], p\u2009=\u20090.0281), PFS (HR 0.65 [95% CI 0.48-0.89]; p\u2009=\u20090.0074) and higher disease control rates (DCR, 86.4% vs 67.5%, p\u2009=\u20090.0096), within the pembrolizumab cohort. No significant associations were found between FHC and OS/PFS/DCR within the chemotherapy cohort. We explored the association between FHC and somatic DNA damage response (DDR) gene alterations as underlying mechanism to our findings in a parallel cohort of 118 NSCLC, 16.9% of whom were FHC-high. The prevalence of\u2009 65\u20091 somatic DDR gene mutation was 20% and 24.5% (p\u2009=\u20090.6684) in FHC-high vs. FHC-low/negative, with no differences in tumor mutational burden (6.0 vs. 7.6 Mut/Mb, p\u2009=\u20090.6018) and tumor cell PD-L1 expression. FHC-high status identifies NSCLC patients with improved outcomes from pembrolizumab but not chemotherapy, independent of somatic DDR gene status. Prospective studies evaluating FHC alongside germline genetic testing are warranted