Citations

Abstract Retinoblastoma (RB) protein can exert tumor suppressor functions even when it becomes phosphorylated. It is thus essential to understand how phosphorylated RB (p-RB) expression and function are regulated. Here, we demonstrated that RING finger domain protein TRIM28 bound and promoted ubiquitination and degradation of CDK4/6-phosphorylated RB protein. SETDB1, a known TRIM28 binding partner, protected p-RB from degradation through the binding of methylated RB by its Tudor domain independent of its methyltransferase activity. SETDB1 was found to be frequently overexpressed due to gene amplification and positively correlated with p-RB in prostate cancer patient specimens. Inhibition of SETDB1 expression using a gene-specific antisense oligonucleotide (ASO) reduced tumor growth but accelerated RB protein degradation, limiting the therapeutic efficacy. However, coadministration of the CDK4/6 inhibitor palbociclib blocked ASO-induced RB degradation and resulted in a much greater cancer-inhibitory effect than each inhibitor alone both in vitro and in vivo. This study identified CDK4/6-dependent, TRIM28-mediated proteasomal degradation as a mechanism of RB inactivation and reveals SETDB1 as a key inhibitor of this process. Our findings suggest that combined targeting of SETDB1 and CDK4/6 represents a viable approach for the treatment of cancers with SETDB1 gene amplification or overexpression. Significance: The identification of a role for TRIM28 and SETDB1 in regulating CDK4/6-phosphorylated RB stability uncovers a combination strategy using CDK4/6 and SETDB1 inhibition to decrease RB degradation and inhibit cancer growth.

Introduction: Fibromuscular dysplasia (FMD) is a non-atherosclerotic arteriopathy of unknown etiology, afflicting mostly middle-aged women. It is characterized by stenotic lesions of the vascular wall in middle-size arteries, sometimes associated with dissection, aneurysm or tortuosity. Renovascular hypertension is a major presentation of FMD and 50–85% of FMD patients are hypertensive. Methods & Results: We report results from the first genome-wide association meta-analysis of six studies including 1556 FMD cases and 7100 controls. We identified four robustly associated loci (PHACTR1, LRP1, ATP2B1, and LIMA1). Using colocalization and transcriptome-wide association analysis with arterial expression datasets, we found PHACTR1, LRP1 and ATP2B1 as target genes at FMD loci and identified one additional locus (SLC24A3). We analysed chromatin accessibility in artery-derived primary cells and found that FMD associated variants were located in arterial specific regulatory elements active in smooth muscle cells. Target genes were broadly involved in mechanisms related to actin cytoskeleton and intracellular calcium homeostasis, central to vascular contraction. All FMD loci were also associated to at least one blood pressure trait. Colocalization analysis supported the same genetic variants to cause association to FMD and systolic blood pressure at PHACTR1, ATP2B1 and LIMA1 loci, and for FMD and pulse pressure at PHACTR1, LRP1 and ATP2B1 loci. Using genome-wide genetic correlation, we found a significant association between FMD and blood pressure. Mendelian Randomization analysis supported genetically determined systolic blood pressure to significantly increase the risk of FMD. However, we confirmed using adjusted and stratified analyses that hypertension status alone was not driving the association with FMD at top loci. We also report an important genetic overlap of FMD with migraine, intracranial aneurysm, coronary artery disease and myocardial infarction. Conclusion: Our study confirms FMD as a polygenic disease following a complex genetic mode of inheritance. We find that FMD shares an important part of its genetic basis with blood pressure regulation, involving in particular several genes related to intracellular calcium homeostasis and vascular smooth muscle contraction. These results are consistent with previous studies showing systemic alterations in vascular contraction in FMD patients, even outside of affected arterial beds. While the mechanisms through which FMD may cause renovascular hypertension are well known, further studies will be required to determine how alterations in vascular contraction may lead to the arterial remodeling observed in FMD.

AbstractBackgroundThe advent of antibiotic-coated devices has reduced the rate of inflatable penile prosthesis (IPP) infections; however, this may have altered microbial profiles when infections do occur.AimTo describe the timing and causative organisms behind infection of infection retardant–coated IPPs in the context of our institution’s perioperative antimicrobial protocols.MethodsWe retrospectively reviewed all patients undergoing IPP placement at our institution from January 2014 to January 2022. In all patients, perioperative antibiotic administration was congruent with American Urological Association guidelines. Boston Scientific devices are impregnated with InhibiZone (rifampin and minocycline), and all Coloplast devices were soaked in rifampin and gentamicin. Intraoperative irrigation was performed with betadine 5% irrigation prior to November 2016 and with vancomycin-gentamicin solution afterward. Cases involving prosthesis infection were identified, and variables were extracted from the medical record. Descriptive and comparative statistics were tabulated to identify clinical characteristics, including patient comorbidities, prophylaxis regimen, symptom onset, and intraoperative culture result. We previously reported an increased infection risk with Betadine irrigation and stratified results accordingly.OutcomesThe primary outcome was time to infectious symptoms, while the secondary outcome was description of device cultures at the time of explantation.ResultsA total of 1071 patients underwent IPP placement over 8 years with an overall infection rate of 2.6% (28/1071). After discontinuation of Betadine, the overall infection rate was significantly lower at 0.9% (8/919) with a relative risk of 16.9 with Betadine (P < .0001). Primary procedures represented 46.4% (13/28). Of 28 patients with infection, only 1 had no identified risk factors; the remainder included Betadine at 71% (20/28), revision/salvage procedure at 53.6% (15/28), and diabetes at 50% (14/28). Median time to symptoms was 36 days (IQR, 26-52); almost 30% of patients had systemic symptoms. Organisms with high virulence, or ability to cause disease, were found in 90.5% (19/21) of positive cultures.Clinical ImplicationsOur study revealed a median time to symptoms of just over 1 month. Risk factors for infection were Betadine 5% irrigation, diabetes, and revision/salvage cases. Over 90% causative organisms were virulent, demonstrating a microbial profile trend since antibiotic coating development.Strengths and LimitationsThe large prospectively maintained database is a strength along with the ability to follow specific changes in perioperative protocols. The retrospective nature of the study is a limitation as well as the low infection rate, which limits certain subanalyses from being performed.ConclusionIPP infections present in a delayed manner despite the rising virulence of infecting organisms. These findings highlight areas for improvement in perioperative protocols in the contemporary prosthetics era.

Abstract The tumor microenvironment (TME) plays a crucial role in mediating the tumor immune response, thereby affecting patient outcomes in follicular lymphoma (FL). Using CyTOF, we analyzed a cohort of 82 FL patients with biopsy specimens collected before the first treatment regimen was administered. Our results showed that there was a difference in the T-cell phenotype in patients with various TMEs. Patients with TMEs rich in monocyte/macrophages/NK cells had more CD8+ cells and tended to have fewer TN, but more TEMRA and TEXH cells. Patients enriched with T cells exhibited a T-cell phenotype skewed toward to TCM and patients with B cell dominance tended to have significant higher number of TFH cells. T-cell phenotypes with more terminally differentiated, senescent and exhausted cells were highly represented in patients with less T cells when compared to patients with more T cells. We observed that CD57+ TFH cells was significantly more abundant in patients who had disease progression than patients who had complete response to therapy, suggesting a role for CD57+ TFH cells in promoting malignant cell growth in FL. Consistent with this finding, we observed that increased numbers of CD57+ TFH cells correlated with an inferior survival in FL. Using CITE-seq technology, we found that CD57+ TFH cells exhibited a substantially different transcriptome from CD57− TFH cells. Genes that were differentially upregulated in CD57+ TFH cells when compared to CD57− TFH cells included genes involved in cell survival, compromised inflammatory response, and metabolic activation. Taken together, our results indicate different tumor microenvironments among patient groups that is associated with variable T-cell phenotypes.

Abstract T cell Ig and ITIM domain (TIGIT) is an immune checkpoint molecule and its ligation delivers an inhibitory signal to T cells that negatively regulates anti-tumor responses. However, the expression and biological relevance of TIGIT in B-cell non-Hodgkin lymphoma (NHL) is completely unknown. To phenotypically characterize TIGIT+ T cell subsets, we profiled TIGIT-expressing CD4+ and CD8+ T cells from biopsy specimens of B-cell NHL. TIGIT expression was not detectable on T cells from peripheral blood, however, TIGIT is constitutively expressed on CD4+ T cells from biopsy specimens of B-cell NHL with a median of 44.8% (range: 25.9–62.2, n=8) of CD4+ T cells expressing TIGIT. By profiling the CD4+TIGIT+ T cells, we observed that CD25 was highly expressed on intratumoral CD4+TIGIT+ T cells. Intracellular staining revealed that Foxp3 was expressed by intratumoral CD4+TIGIT+ T cells. These intratumoral CD4+TIGIT+ T cells expressed high level of chemokine receptor CCR4 and were absent of IL-7 receptor-α (CD127). Functionally, CD4+TIGIT+ T cells displayed reduced cytokine production, as the number of IFN-γ- and TNF-α-producing cells was lower in the TIGIT+ population than in TIGIT− T cells. While TIGIT expression can be induced by TCR activation, treatment of T cells with IL-2 and TGF-β inhibited TCR-mediated TIGIT induction. Furthermore, lymphoma B cells were involved in TIGIT induction as in vitro depletion of lymphoma B cells altered upregulation of TIGIT on CD4+ T cells in B-cell NHL. Taken together, these results indicated that constitutive expression of TIGIT defines a CD4+ population with similar phenotype to regulatory T cells. Inhibition of TIGIT signaling may be an additional mechanism to prevent T-cell suppression in B-cell NHL

Abstract Severe influenza A virus infection typically triggers excessive and detrimental lung inflammation with massive cell infiltration and hyper-production of cytokines and chemokines. Defining research models to alleviate lung damage without compromising viral control is critical. We identified a novel function for nuclear matrix protein 4 (NMP4), a zinc-finger-containing transcription factor in regulating lung inflammation and antiviral innate immune responses. Nmp4-deficient mice are protected from H1N1 influenza infection, losing less body weight compared to the wild-type mice. Nmp4-deficiency does not affect viral clearance or adaptive immune responses, but significantly reduces the accumulation of neutrophils and monocytes in the lungs and the early inflammation. Consistent with fewer innate cells in the airways, infected Nmp4-deficient mice show decreased expression of chemokine genes Ccl2 and Cxcl1 as well as pro-inflammatory cytokine genes Il1b and Il6. We further reveal that NMP4 binds to the promoters and/or conserved non-coding sequences (CNS) of Ccl2 and Cxcl1 in mouse lung epithelial cells. Besides, the knockdown of NMP4 by CRISPR/Cas9 or siRNA significantly lessens the luciferase activities of Ccl2 and Cxcl1 promoters and/or CNS. Taken together, our data suggest that NMP4 regulates monocyte- and neutrophil-attracting chemokine expression in airway epithelial cells upon influenza infection, resulting in exaggerated innate inflammation and lung tissue damage.

Abstract Although extensively studied, the exact mechanism by which TGF-β mediates T-cell inhibition is not entirely clear. When we examined the effect of TGF-β on the phenotype and function of T cells, we noted that CD70 was significantly up-regulated by TGF-β. This surprising finding led us to explore the underlying mechanism by which TGF-β upregulates CD70 expression on T cells and to define the function of CD70+ cells in B-cell NHL. Interestingly, we observed that TGF-β preferentially induced CD70 expression on effector memory T cells while it predominantly up-regulated Foxp3 on naïve T cells. CD70 induction is Smad3-dependent and involves Stat5 signaling. CD70+ T cells were highly susceptible to TGF-β-mediated apoptosis and that transfection of Jurkat cells with the CD70 gene led to a significantly reduced survival. TGF-β-induced CD70+ T cells had a diminished response to immune stimulation associated with downregulation of co-stimulatory molecules CD27 and CD28. CD70-expressing T cells from biopsy specimens of patients with B-cell NHL failed to produce cytokines and displayed no signal transduction when stimulated. CD70-expressing T cells were highly represented in follicular NHL biopsy specimens and increased numbers of CD70+ cells correlated with an inferior patient outcome. These novel findings not only revealed a biological role for a TGF-β/CD70 axis in mediating immune inhibition, but also may lead to therapeutic approaches in patients with B-cell NHL.

Objective: Sleep fragmentation determined by repetitive arousals from sleep in obstructive sleep apnea (OSA) is associated with hypertension. We aimed to quantify the independent association of arousals during rapid eye movement (REM)/non-rapid eye movement (NREM) sleep with prevalent hypertension. Methods: We included adults with 4 h of total sleep time and at least 30 min of REM sleep obtained from overnight in-laboratory polysomnography. Logistic regression models were fitted to explore the association between arousals during REM/NREM sleep and prevalent hypertension. All models controlled for OSA metrics and arousals during NREM/REM sleep, either by statistical adjustment or by stratification. Results: The sample comprised of 11 643 patients, of which 10 055 were OSA patients. Fully adjusted models demonstrated significant dose-relationships between arousal index during REM sleep (AI-REM) and prevalent hypertension (P trend = 0.002). The higher relative odds of prevalent hypertension were most evident with AI-REM > 40 events/h. In OSA patients with arousal index during NREM sleep (AI-NREM) <15 events/h, every10-unit increase in the AI-REM was associated with 18% higher odds of hypertension (odds ratio, 1.18; 95% confidence interval, 1.11–1.27) in OSA. On the contrary, AI-NREM was not a significant predictor of hypertension in any of the models. Conclusions: Our findings indicate that arousals during REM sleep are associated with prevalent hypertension. This is clinically relevant because treatment of OSA is often limited to the first half of the sleep period leaving most of sleep fragmentation during REM sleep untreated.