Selective transcription and cellular proliferation induced by PDGF require histone deacetylase activity

Histone deacetylases (HDACs) are key regulatory enzymes involved in the control of gene expression and their inhibition by specific drugs has been widely correlated to cell cycle arrest, terminal differentiation, and apoptosis. Here, we investigated whether HDAC activ- ity was required for PDGF-dependent signal transduction and cellular proliferation. Exposure of PDGF-stimulated NIH3T3 fibroblasts to the HDAC inhibitor trichostatin A (TSA) potently repressed the expression of a group of genes correlated to PDGF-dependent cel- lular growth and pro-survival activity. Moreover, we show that TSA interfered with STAT3-dependent transcriptional activity induced by PDGF. Still, neither phosphorylation nor nuclear translocation and DNA-binding in vitro and in vivo of STAT3 were affected by using TSA to interfere with PDGF stimulation. Finally, TSA treatment resulted in the suppression of PDGF-dependent cellular prolif- eration without affecting cellular survival of NIH3T3 cells. Our data indicate that inhibition of HDAC activity antagonizes the mitogenic effect of PDGF, suggesting that these drugs may specifically act on the expression of STAT-dependent, PDGF-responsive genes

Activation of the Erk8 Mitogen-activated Protein (MAP) Kinase by RET/PTC3, a Constitutively Active Form of the RET Proto-oncogene

Mitogen-activated protein (MAP) kinases have a central role in several biological functions, including cell adhesion and spreading, chemotaxis, cell cycle progression, differentiation, and apoptosis. Extracellular signal-regulated kinase 8 (Erk8) is a large MAP kinase whose activity is controlled by serum and the c-Src non-receptor tyrosine kinase. Here, we show that RET/PTC3, an activated form of the RET proto-oncogene, was able to activate Erk8, and we demon- strate that such MAP kinase participated in RET/PTC3-dependent stimulation of the c-jun promoter. By using RET/PTC3 molecules mutated in specific tyrosine autophosphorylation sites, we charac- terized Tyr981, a known binding site for c-Src, as a major determi- nant of RET/PTC3-induced Erk8 activation, although, surprisingly, the underlying mechanism did not strictly depend on the activity of Src. In contrast, we present evidence that RET/PTC3 acts on Erk8 through Tyr981-mediated activation of c-Abl. Furthermore, we localized the region responsible for the modulation of Erk8 activity by the RET/PTC3 and Abl oncogenes in the Erk8 C-terminal domain. Altogether, these results support a role for Erk8 as a novel effector of RET/PTC3 and, therefore, RET biological functions

The Platelet-derived Growth Factor Controls c-myc Expression through a JNK- and AP-1-dependent Signaling Pathway *

Pro-inflammatory cytokines, environmental stresses, as well as receptor tyrosine kinases regulate the activity of JNK. In turn, JNK phosphorylates Jun members of the AP-1 family of transcription factors, thereby controlling processes as different as cell growth, differentiation, and apoptosis. Still, very few targets of the JNK-Jun pathway have been identified. Here we show that JNK is required for the induction of c-myc expression by PDGF. Furthermore, we identify a phylogenetically conserved AP-1-responsive element in the promoter of the c-myc proto-oncogene that recruits in vivo the c-Jun and JunD AP-1 family members and controls the PDGF-dependent transactivation of the c-myc promoter. These findings suggest the existence of a novel biochemical route linking tyrosine kinase receptors, such as those for PDGF, and c-myc expression through JNK activation of AP-1 transcription factors. They also provide a novel potential mechanism by which both JNK and Jun proteins may exert either their proliferative or apoptotic potential by stimulating the expression of the c-myc proto-oncogene

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population

Defining Kawasaki disease and pediatric inflammatory multisystem syndrome-temporally associated to SARS-CoV-2 infection during SARS-CoV-2 epidemic in Italy: results from a national, multicenter survey

Background: There is mounting evidence on the existence of a Pediatric Inflammatory Multisystem Syndrome-temporally associated to SARS-CoV-2 infection (PIMS-TS), sharing similarities with Kawasaki Disease (KD). The main outcome of the study were to better characterize the clinical features and the treatment response of PIMS-TS and to explore its relationship with KD determining whether KD and PIMS are two distinct entities. Methods: The Rheumatology Study Group of the Italian Pediatric Society launched a survey to enroll patients diagnosed with KD (Kawasaki Disease Group - KDG) or KD-like (Kawacovid Group - KCG) disease between February 1st 2020, and May 31st 2020. Demographic, clinical, laboratory data, treatment information, and patients' outcome were collected in an online anonymized database (RedCAP®). Relationship between clinical presentation and SARS-CoV-2 infection was also taken into account. Moreover, clinical characteristics of KDG during SARS-CoV-2 epidemic (KDG-CoV2) were compared to Kawasaki Disease patients (KDG-Historical) seen in three different Italian tertiary pediatric hospitals (Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste; AOU Meyer, Florence; IRCCS Istituto Giannina Gaslini, Genoa) from January 1st 2000 to December 31st 2019. Chi square test or exact Fisher test and non-parametric Wilcoxon Mann-Whitney test were used to study differences between two groups. Results: One-hundred-forty-nine cases were enrolled, (96 KDG and 53 KCG). KCG children were significantly older and presented more frequently from gastrointestinal and respiratory involvement. Cardiac involvement was more common in KCG, with 60,4% of patients with myocarditis. 37,8% of patients among KCG presented hypotension/non-cardiogenic shock. Coronary artery abnormalities (CAA) were more common in the KDG. The risk of ICU admission were higher in KCG. Lymphopenia, higher CRP levels, elevated ferritin and troponin-T characterized KCG. KDG received more frequently immunoglobulins (IVIG) and acetylsalicylic acid (ASA) (81,3% vs 66%; p = 0.04 and 71,9% vs 43,4%; p = 0.001 respectively) as KCG more often received glucocorticoids (56,6% vs 14,6%; p < 0.0001). SARS-CoV-2 assay more often resulted positive in KCG than in KDG (75,5% vs 20%; p < 0.0001). Short-term follow data showed minor complications. Comparing KDG with a KD-Historical Italian cohort (598 patients), no statistical difference was found in terms of clinical manifestations and laboratory data. Conclusion: Our study suggests that SARS-CoV-2 infection might determine two distinct inflammatory diseases in children: KD and PIMS-TS. Older age at onset and clinical peculiarities like the occurrence of myocarditis characterize this multi-inflammatory syndrome. Our patients had an optimal response to treatments and a good outcome, with few complications and no deaths

Selective transcription and cellular proliferation induced by PDGF require histone deacetylase activity

Histone deacetylases (HDACs) are key regulatory enzymes involved in the control of gene expression in any cell type, and their inhibition by specific drugs has been widely correlated to cell cycle arrest, terminal differentiation and apoptosis. Although some of these compounds gave already very promising results in Phase I and II clinical trials for a large array of tumors, the molecular targets responsible for their anti-tumoral activity are still largely unknown. Here, we investigated whether HDAC activity was required for PDGF-dependent signal transduction and cellular proliferation. Exposure of PDGF-stimulated NIH3T3 fibroblasts to the HDAC inhibitor trichostatin A (TSA) potently repressed the expression of a group of genes strongly correlated to PDGF-dependent cellular growth and pro-survival activity, namely c-myc, VEGF and bcl-XL. Moreover, we show that TSA interfered with STAT3-dependent transcriptional activity induced by PDGF, suggesting STAT proteins as mediators of HDAC activity on PDGF transcriptional responses. Still, neither phosphorylation nor nuclear translocation and DNA-binding of STAT3 were affected by using TSA to interfere with PDGF stimulation. Finally, TSA treatment resulted in the suppression of PDGF-dependent cell proliferation as scored by a bromodeoxyuridine (BrdU) incorporation assay. Our data indicate that inhibition of HDAC activity by specific pharmacological inhibitors antagonizes the mitogenic effect of PDGF, suggesting that these drugs may specifically act on the expression of growth-related, STAT-dependent, PDGF early- and late-responsive genes

Identification of Inquilinus limosus in Cystic Fibrosis: a first report in Italy

Cystic fibrosis is a genetic disorder associated with a polymicrobial lung infection where classical pathogens and newly identified bacteria may interact. Inquilinus limosus is an a-proteobacterium recently isolated in the airways of cystic fibrosis patient. We report the first case in Italy of I.limosus isolation from the sputum sample of a cystic fibrosis patient. The patient is a 20-years-old man with cystic fibrosis, regularly attending the Regional Care Center for Cystic Fibrosis at the Federico II University Hospital of Naples. Microbiological culture methods detected a mucoid gram negative bacillus in the patient???s sputum sample. The isolate exhibited a distinct antimicrobial susceptibility profile with a high MIC for several drugs. The MALDI-TOF mass spectrometry analysis indicated the bacterium isolated as I. limosus, confirmed by 16s rDNA sequence analysis. The described clinical case demonstrates how the bacterial biodiversity in the airways of cystic fibrosis patients is still underestimated. Cystic fibrosis lung represents an ecological niche suitable for growth of a wide variety of unusual bacteria not commonly associated with human diseases, such as I. limosus. Therefore further studies are needed to evaluate the epidemiology and clinical implications of I. limosus in the physiopathology of cystic fibrosis lung infection

TYROSINE KINASE INHIBITORS DISCONTINUATION IN CHRONIC MYELOID LEUKEMIA: A RETROSPECTIVE ANALYSIS OF 208 ITALIAN PATIENTS

Background: In the last 10 years different studies analyzed the outcome of patients (pts) with sustained complete molecular remission (CMR) who discontinued imatinib, reporting rates of treatment-free remission (TFR) ranging from 33 to 66% depending on the definition of molecular relapse. On these bases it is judged safe to discontinue treatment with tyrosine-kinase inhibitors (TKIs) in experimental contexts. Aims: To evaluate TFR in the setting of clinical practice according to the Italian experience where most of the pts who discontinued TKIs were not included in prospective protocols. Methods: We retrospectively analyzed the outcome of pts treated in 23 Divisions of Hematology in Italy, who discontinued TKIs in deep molecular response (MR). General and clinical information such as TKI at discontinuation, line of treatment, type of MR at discontinuation, reasons for discontinuation were collected. We estimated TFR with the Kaplan-Meier method. Prognostic factors for TFR were assessed by univariate Cox regression model analysis. Results:We analyzed a total of 208 pts who discontinued TKIs from June 2003 to October 2015. Median age was 59 years (Interquartile Range, IQR, 46-72). 102 were male, 106 female; 52%, 35% and 13% were low, intermediate and high Sokal score respectively. 168 pts (81%) discontinued in first line; 38 pts in second line (63% for intolerance to prior TKIs) and 2 pts in third line. 153 pts (74%) were on treatment with imatinib (all frontline), 26% with either nilotinib or dasatinib. Median duration of treatment with the last TKI was 75.2 mos (IQR 50-114); median time to CMR (undetectable transcript or MR4/MR4.5/MR5) with the last TKI was 23.3 mos (IQR 11.1-45.2). Median duration of CMR was 23 mos (IQR 11-45) before stop. At 3 mos of last TKI 28% of pts were in MR3, 26% were in PCyR and/or had a transcript <10%, 45% were in CCyR and/or had a transcript <1%, and 1% had no response. 184 pts had a response defined according to molecular standardization: 8% were MR3, 30% were MR4, 36% were MR4.5, 26% were MR5. Reasons for discontinuation were: toxicity for 28% of pts, pregnancy for 10%, pt request for 56%, enrollment in ISAV protocol for 10 pts. After a median follow-up of 11 mos (IQR 1-149), estimated TFR was 71% (95%CI 64.6%>78.1%) (figure 1). 69 pts restarted treatment. Reasons for restarting were: loss of MR4 for 20% of pts, loss of MR3 for 55%, loss of CCyR for 12%, other reasons for 13%. Median time to restart treatment was 6 mos (IQR 4-11). We assessed age, sex, Sokal score, type of transcript, previous IFN therapy, duration of TKI therapy, response at 3 mos, time to CMR, CMR duration, line of therapy at stop, depth of MR, reasons for stop as potential prognostic factors for TFR, but no statistically significant association were found, with the exception of response MR5 at stop (MR5 vs MR4, HR: 0.43, 95%CI 0.21-0.87, p=0.02) and age [HR 0.62 (95%CI 0.42-0.93, p=0.02), i.e. a decreased risk in older vs younger pts (difference of age=26 years)]. Pts who had to restart therapy were treated with imatinib (57), nilotinib (10), dasatinib (2). All of them regained at least MR3. No pts progressed. All pts were alive at the last follow up with the exception of 7 who died for reasons unrelated to CML

TYROSINE KINASE INHIBITORS DISCONTINUATION IN CHRONIC MYELOID LEUKEMIA: A RETROSPECTIVE ANALYSIS OF 208 ITALIAN PATIENTS

Background: In the last 10 years different studies analyzed the outcome of patients (pts) with sustained complete molecular remission (CMR) who discontinued imatinib, reporting rates of treatment-free remission (TFR) ranging from 33 to 66% depending on the definition of molecular relapse. On these bases it is judged safe to discontinue treatment with tyrosine-kinase inhibitors (TKIs) in experimental contexts. Aims: To evaluate TFR in the setting of clinical practice according to the Italian experience where most of the pts who discontinued TKIs were not included in prospective protocols. Methods: We retrospectively analyzed the outcome of pts treated in 23 Divisions of Hematology in Italy, who discontinued TKIs in deep molecular response (MR). General and clinical information such as TKI at discontinuation, line of treatment, type of MR at discontinuation, reasons for discontinuation were collected. We estimated TFR with the Kaplan-Meier method. Prognostic factors for TFR were assessed by univariate Cox regression model analysis. Results:We analyzed a total of 208 pts who discontinued TKIs from June 2003 to October 2015. Median age was 59 years (Interquartile Range, IQR, 46-72). 102 were male, 106 female; 52%, 35% and 13% were low, intermediate and high Sokal score respectively. 168 pts (81%) discontinued in first line; 38 pts in second line (63% for intolerance to prior TKIs) and 2 pts in third line. 153 pts (74%) were on treatment with imatinib (all frontline), 26% with either nilotinib or dasatinib. Median duration of treatment with the last TKI was 75.2 mos (IQR 50-114); median time to CMR (undetectable transcript or MR4/MR4.5/MR5) with the last TKI was 23.3 mos (IQR 11.1-45.2). Median duration of CMR was 23 mos (IQR 11-45) before stop. At 3 mos of last TKI 28% of pts were in MR3, 26% were in PCyR and/or had a transcript <10%, 45% were in CCyR and/or had a transcript <1%, and 1% had no response. 184 pts had a response defined according to molecular standardization: 8% were MR3, 30% were MR4, 36% were MR4.5, 26% were MR5. Reasons for discontinuation were: toxicity for 28% of pts, pregnancy for 10%, pt request for 56%, enrollment in ISAV protocol for 10 pts. After a median follow-up of 11 mos (IQR 1-149), estimated TFR was 71% (95%CI 64.6%>78.1%) (figure 1). 69 pts restarted treatment. Reasons for restarting were: loss of MR4 for 20% of pts, loss of MR3 for 55%, loss of CCyR for 12%, other reasons for 13%. Median time to restart treatment was 6 mos (IQR 4-11). We assessed age, sex, Sokal score, type of transcript, previous IFN therapy, duration of TKI therapy, response at 3 mos, time to CMR, CMR duration, line of therapy at stop, depth of MR, reasons for stop as potential prognostic factors for TFR, but no statistically significant association were found, with the exception of response MR5 at stop (MR5 vs MR4, HR: 0.43, 95%CI 0.21-0.87, p=0.02) and age [HR 0.62 (95%CI 0.42-0.93, p=0.02), i.e. a decreased risk in older vs younger pts (difference of age=26 years)]. Pts who had to restart therapy were treated with imatinib (57), nilotinib (10), dasatinib (2). All of them regained at least MR3. No pts progressed. All pts were alive at the last follow up with the exception of 7 who died for reasons unrelated to CML