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Background: Rare diseases are chronic and life-threatening disorders affecting < 1 person every 2,000. For most of them, clinical symptoms and signs can be observed at birth or childhood. Approximately 80% of all rare diseases have a genetic background and most of them are monogenic conditions. In addition, while the majority of these diseases is still incurable, early diagnosis and specific treatment can improve patients' quality of life. Transplantation is among the therapeutic options and represents the definitive treatment for end-stage organ failure, both in children and adults. The aim of this paper was to analyze, in a large cohort of Italian patients, the main rare genetic diseases that led to organ transplantation, specifically pointing the attention on the pediatric cohort. Results: To the purpose of our analysis, we considered heart, lung, liver and kidney transplants included in the Transplant Registry (TR) of the Italian National Transplantation Center in the 2002–2019 timeframe. Overall, 49,404 recipients were enrolled in the cohort, 5.1% of whom in the pediatric age. For 40,909 (82.8%) transplant recipients, a disease diagnosis was available, of which 38,615 in the adult cohort, while 8,495 patients (17.2%) were undiagnosed. There were 128 disease categories, and of these, 117 were listed in the main rare disease databases. In the pediatric cohort, 2,294 (5.6%) patients had a disease diagnosis: of the 2,126 (92.7%) patients affected by a rare disease, 1,402 (61.1%) presented with a monogenic condition. As expected, the frequencies of pathologies leading to organ failure were different between the pediatric and the adult cohort. Moreover, the pediatric group was characterized, compared to the adult one, by an overall better survival of the graft at ten years after transplant, with the only exception of lung transplants. When comparing survival considering rare vs non-rare diseases or rare and monogenic vs rare non-monogenic conditions, no differences were highlighted for kidney and lung transplants, while rare diseases had a better survival in liver as opposed to heart transplants. Conclusions: This work represents the first national survey analyzing the main genetic causes and frequencies of rare and/or monogenic diseases leading to organ failure and requiring transplantation both in adults and children. [ABSTRACT FROM AUTHOR]

Background: Rare diseases are chronic and life-threatening disorders affecting < 1 person every 2,000. For most of them, clinical symptoms and signs can be observed at birth or childhood. Approximately 80% of all rare diseases have a genetic background and most of them are monogenic conditions. In addition, while the majority of these diseases is still incurable, early diagnosis and specific treatment can improve patients' quality of life. Transplantation is among the therapeutic options and represents the definitive treatment for end-stage organ failure, both in children and adults. The aim of this paper was to analyze, in a large cohort of Italian patients, the main rare genetic diseases that led to organ transplantation, specifically pointing the attention on the pediatric cohort.Results: To the purpose of our analysis, we considered heart, lung, liver and kidney transplants included in the Transplant Registry (TR) of the Italian National Transplantation Center in the 2002-2019 timeframe. Overall, 49,404 recipients were enrolled in the cohort, 5.1% of whom in the pediatric age. For 40,909 (82.8%) transplant recipients, a disease diagnosis was available, of which 38,615 in the adult cohort, while 8,495 patients (17.2%) were undiagnosed. There were 128 disease categories, and of these, 117 were listed in the main rare disease databases. In the pediatric cohort, 2,294 (5.6%) patients had a disease diagnosis: of the 2,126 (92.7%) patients affected by a rare disease, 1,402 (61.1%) presented with a monogenic condition. As expected, the frequencies of pathologies leading to organ failure were different between the pediatric and the adult cohort. Moreover, the pediatric group was characterized, compared to the adult one, by an overall better survival of the graft at ten years after transplant, with the only exception of lung transplants. When comparing survival considering rare vs non-rare diseases or rare and monogenic vs rare non-monogenic conditions, no differences were highlighted for kidney and lung transplants, while rare diseases had a better survival in liver as opposed to heart transplants.Conclusions: This work represents the first national survey analyzing the main genetic causes and frequencies of rare and/or monogenic diseases leading to organ failure and requiring transplantation both in adults and children. [ABSTRACT FROM AUTHOR]

Targeting adenosine triphosphate (ATP) metabolism and adenosinergic signaling in cancer is gaining momentum, as increasing evidence is showing their relevance in tumor immunology and biology. Chronic lymphocytic leukemia (CLL) results from the expansion of a population of mature B cells that progressively occupies the bone marrow (BM), the blood, and peripheral lymphoid organs. Notwithstanding significant progress in the treatment of these patients, the cure remains an unmet clinical need, suggesting that novel drugs or drug combinations are needed. A unique feature of CLL is its reliance on micro-environmental signals for proliferation and cell survival. We and others have shown that the lymphoid niche, an area of intense interactions between leukemic and bystander non-tumor cells, is a typically hypoxic environment. Here adenosine is generated by leukemic cells, as well as by cells of myeloid origin, acting through autocrine and paracrine mechanisms, ultimately affecting tumor growth, limiting drug responses, and skewing the immune cells towards a tolerant phenotype. Hence, understanding the mechanisms through which this complex network of enzymes, receptors, and metabolites functions in CLL, will pave the way to the use of pharmacological agents targeting the system, which, in combination with drugs targeting leukemic cells, may get us one step closer to curing these patients. [ABSTRACT FROM AUTHOR]

Abstract: Chronic lymphocytic leukemia (CLL) cells display features consistent with a defect in apoptosis and exhibit prolonged survival in vivo. Survival of these malignant cells is influenced by interactions with non-leukemic cells located in permissive niches in lymphoid organs. Leukemic cells subvert the normal architecture of the lymphoid organs, recruiting stromal cells, dendritic cells and T lymphocytes, all reported as playing active roles in the survival and proliferation of CLL. The same survival-promoting environment also rescues/protects leukemic cells from cytotoxic therapies, giving way to disease relapse. This review summarizes and discusses current knowledge about the intricate network of soluble and cell-bound signals regulating the life and death of CLL cells in different districts. At the same time, it seeks to hone in on which discrete molecular elements are best suited as targets for treating this still incurable disease. [Copyright &y& Elsevier]

Because of its relatively indolent clinical course, chronic lymphocytic leukemia (CLL) offers a versatile model for testing novel therapeutic regimens and drug combinations. Nicotinamide is the main NAD +precursor and a direct inhibitor of four classes of enzymes, including the sirtuins. SIRT1, the main member of the sirtuin family, inactivates p53 by deacetylating a critical lysine residue. In this study, we showed that CLL cells express high levels of functional SIRT1, which is inhibited by exogenous nicotinamide. This agent blocks proliferation and promotes apoptosis selectively in leukemic cells that express wild-type (wt) p53. Nicotinamide modulates the p53-dependent genes p21, NOXA, BAX, and Mcl-1, indicating an activation of the p53 pathway and of caspase-3. DNA-damaging chemotherapeutics, such as etoposide, activate a functional loop linking SIRT1 and p53 through the induction of miR-34a. When leukemic cells are simultaneously exposed to nicotinamide and etoposide, we observe a significant increase in miR-34a levels with a concomitant inhibition of SIRT1. Furthermore, p53 acetylation levels are higher than with either agent used alone. Overall, treatment with both nicotinamde and etoposide shows strongly synergistic effects in the induction of apoptosis. We therefore concluded that nicotinamide has the dual property of inhibiting SIRT1 through a noncompetitive enzymatic block (p53 independent) and at the same time through miR-34a induction (p53 dependent). These observations suggested the therapeutic potential of nicotinamide, a novel, safe, and inexpensive drug, to be used in addition to chemotherapy for CLL patients with wt p53. Cancer Res; 71(13); 4473-83. ©2011 AACR. [ABSTRACT FROM AUTHOR]

Abstract: Nicotinamide adenine dinucleotide (NAD+) is an essential co-enzyme that can be released in the extracellular milieu. Here, it may elicit signals through binding purinergic receptors. Alternatively, NAD+ may be dismantled to adenosine, up-taken by cells and transformed to reconstitute the intracellular nucleotide pool. An articulated ecto-enzyme network is responsible for the nucleotide–nucleoside conversion. CD38 is the main mammalian enzyme that hydrolyzes NAD+, generating Ca2+-active metabolites. Evidence suggests that this extracellular network may be altered or used by tumor cells to (i) nestle in protected areas, and (ii) evade the immune response. We have exploited chronic lymphocytic leukemia as a model to test the role of the ecto-enzyme network, starting by analyzing the individual elements that make up the whole picture. [Copyright &y& Elsevier]

Abstract: CLL is characterized by a dynamic balance between cells proliferating in the lymphoid organs and circulating cells resisting programmed cell death. Regulating this equilibrium entails complex interactions between tumor and host, modulated by a set of surface molecules expressed by the CLL cell according to environmental conditions. The result is a constantly shifting pattern of resistance, apoptosis and proliferation. The CD38 surface molecule is an independent negative prognostic factor expressed by approximately one-third of CLL patients. Our view is that CD38 is crucial to tumor–host communication and that its signals are detrimental to clinical outcome. CD38+ CLL cells can proliferate in vitro in the presence of anti-CD38 mAbs and IL-2 and are more sensitive to the effects of the CXCL12 chemokine. Blockage of CD38 signals impairs CLL cell movement from blood to lymphoid organs, as confirmed using animal models. One model to be explored considers CD38 a key component of the CLL invadosome, a still hypothetical membrane domain containing adhesion molecules, chemokine receptors and matrix metalloproteases. Some components of the invadosome are genetically polymorphic, explaining heterogeneity in functional response. The CD38 gene shows genetic differences in the promoter region, some of which represent an independent risk for Richter transformation. In addition to driving the clinical outcome of the disease, CD38 is thus an excellent candidate therapeutic target for a significant subset of CLL patients. [ABSTRACT FROM AUTHOR]

Circulating cell-free DNA (cfDNA) refers to small fragments of DNA molecules released after programmed cell death and necrosis in several body fluids such as blood, saliva, urine, and cerebrospinal fluid. The discovery of cfDNA has revolutionized the field of non-invasive diagnostics in the oncologic field, in prenatal testing, and in organ transplantation. Despite the potential of cfDNA and the solid results published in the recent literature, several challenges remain, represented by a low abundance, a need for highly sensitive assays, and analytical issues. In this review, the main technical advances in cfDNA analysis are presented and discussed, with a comprehensive examination of the current available methodologies applied in each field. Considering the potential advantages of cfDNA, this biomarker is increasing its consensus among clinicians, as it allows us to monitor patients' conditions in an easy and non-invasive way, offering a more personalized care. Nevertheless, cfDNA analysis is still considered a diagnostic marker to be further validated, and very few centers are implementing its analysis in routine diagnostics. As technical improvements are enhancing the performances of cfDNA analysis, its application will transversally improve patients' quality of life. [ABSTRACT FROM AUTHOR]

Objective The calcium-sensing receptor (CASR) gene encodes a G protein–coupled receptor crucial for calcium homeostasis. Gain-of-function CASR variants result in hypocalcemia, while loss-of-function variants lead to hypercalcemia. This study aims to assess the functional consequences of the novel nonsense CASR variant [c.2897_2898insCTGA, p.(Gln967*) (Q967*)] identified in adolescent patient with chronic hypocalcemia, a phenotype expected for a gain-of-function variants. Design and methods To functionally characterize the Q967* mutant receptor, both wild-type (WT) and mutant CASR were transiently transfected into HEK293T cells and calcium-sensing receptor (CaSR) protein expression and functions were comparatively evaluated using multiple read-outs. Results Western blot analysis revealed that the CaSR mutant protein displayed a lower molecular weight compared with the WT, consistent with the loss of the last 122 amino acids in the intracellular domain. Mitogen-activated protein kinase activation and serum responsive element luciferase assays demonstrated that the mutant receptor had higher baseline activity than the WT. Extracellular-signal-regulated kinase/c-Jun N-terminal kinase phosphorylation, however, remained consistently high in the mutant, without significant modulations following exposure to increasing extracellular calcium (Ca2+o) levels, suggesting that the mutant receptor is more sensitive to Ca2+o compared with the WT. Conclusions This study provides functional validation of the pathogenicity of a novel nonsense CASR variant, resulting in an abnormally hyperfunctioning protein consistent with the patient's phenotype. Functional analyses indicate that mutant receptor is constitutively active and poorly sensitive to increasing concentrations of extracellular calcium, suggesting that the cytoplasmic tail may contain elements regulating signal transduction. [ABSTRACT FROM AUTHOR]

Background: In 2018, our center started a program to offer genetic diagnosis to patients with kidney and liver monogenic rare conditions, potentially eligible for organ transplantation. We exploited a clinical exome sequencing approach, followed by analyses of in silico gene panels tailored to clinical suspicions, obtaining detection rates in line with what reported in literature. However, a percentage of patients remains without a definitive genetic diagnosis. This work aims to evaluate the utility of NGS data re-analysis for those patients with an inconclusive or negative genetic test at the time of first analysis considering that (i) the advance of alignment and variant calling processes progressively improve the detection rate, limiting false positives and false negatives; (ii) gene panels are periodically updated and (iii) variant annotation may change over time. Methods: 114 patients, recruited between 2018 and 2020, with an inconclusive or negative NGS report at the time of first analysis, were included in the study. Re-alignment and variant calling of previously generated sequencing raw data were performed using the GenomSys Variant Analyzer software. Results: 21 previously not reported potentially causative variants were identified in 20 patients. In most cases (n = 19), causal variants were retrieved out of the re-classification from likely benign to variants of unknown significance (VUS). In one case, the variant was included because of inclusion in the analysis of a newly disease-associated gene, not present in the original gene panel, and in another one due to the improved data alignment process. Whenever possible, variants were validated with Sanger sequencing and family segregation studies. As of now, 16 out of 20 patients have been analyzed and variants confirmed in 8 patients. Specifically, in two pediatric patients, causative variants were de novo mutations while in the others, the variant was present also in other affected relatives. In the remaining patients, variants were present also in non-affected parents, raising questions on their re-classification. Conclusions: Overall, these data indicate that periodic and systematic re-analysis of negative or inconclusive NGS data reports can lead to new variant identification or reclassification in a small but significant proportion of cases, with benefits for patients' management. [ABSTRACT FROM AUTHOR]

Despite recent relevant therapeutic progresses, chronic lymphocytic leukemia (CLL) remains an incurable disease. Selinexor, an oral inhibitor of the nuclear export protein XPO1, is active as single agent in different hematologic malignancies, including CLL. The purpose of this study was to evaluate the anti-tumor effects of selinexor, used in combination with chemotherapy drugs (i.e. fludarabine and bendamustine) or with the PI3Kδ inhibitor idelalisib in CLL. Our results showed a significant decrease in CLL cell viability after treatment with selinexor-containing drug combinations compared to each single compound, with demonstration of synergistic cytotoxic effects. Interestingly, this drug synergism was exerted also in the presence of the protective effect of stromal cells. From the molecular standpoint, the synergistic cytotoxic activity of selinexor plus idelalisib was associated with increased regulatory effects of this drug combination on the tumor suppressors FOXO3A and IkBα compared to each single compound. Finally, selinexor was also effective in potentiating the in vivo anti-tumor effects of the PI3Kδ inhibitor in mice treated with the drug combination compared to single agents. Our data provide preclinical evidence of the synergism and potential efficacy of a combination treatment targeting XPO1 and PI3Kδ in CLL. [ABSTRACT FROM AUTHOR]

Despite recent relevant therapeutic progresses, chronic lymphocytic leukemia (CLL) remains an incurable disease. Selinexor, an oral inhibitor of the nuclear export protein XPO1, is active as single agent in different hematologic malignancies, including CLL. The purpose of this study was to evaluate the anti-tumor effects of selinexor, used in combination with chemotherapy drugs (i.e. fludarabine and bendamustine) or with the PI3Kδ inhibitor idelalisib in CLL. Our results showed a significant decrease in CLL cell viability after treatment with selinexor-containing drug combinations compared to each single compound, with demonstration of synergistic cytotoxic effects. Interestingly, this drug synergism was exerted also in the presence of the protective effect of stromal cells. From the molecular standpoint, the synergistic cytotoxic activity of selinexor plus idelalisib was associated with increased regulatory effects of this drug combination on the tumor suppressors FOXO3A and IkBα compared to each single compound. Finally, selinexor was also effective in potentiating the in vivo anti-tumor effects of the PI3Kδ inhibitor in mice treated with the drug combination compared to single agents. Our data provide preclinical evidence of the synergism and potential efficacy of a combination treatment targeting XPO1 and PI3Kδ in CLL. [ABSTRACT FROM AUTHOR]

Host genetic variability contributes to susceptibility to SARS‐CoV‐2 infection and COVID‐19 evolution and the role of HLA system has not clearly emerged, suggesting the involvement of other factors. Studying response to vaccination with Spyke protein mRNA represents an ideal model to highlight whether the humoral or cellular responses are influenced by HLA. Four hundred and sixteen workers, vaccinated with Comirnaty beginning 2021, were selected within the Azienda Ospedaliera Universitaria "Città della Salute e della Scienza di Torino." The humoral response was determined with the LIAISON® kit, while the analysis of the cellular response was performed with the Quantiferon SARS‐CoV‐2 assay, for the S1 (receptor‐binding domain; Ag1) and S1 and S2 (Ag2) subunits of the Spyke protein. Six HLA loci were typed by next‐generation sequencing. Associations between HLA and vaccine response were performed with univariate and multivariate analyses. An association was found between A*03:01, B*40:02 and DPB1*06:01 and high antibody concentration and between A*24:02, B*08:01 and C*07:01 and low humoral responses. The haplotype HLA‐A*01:01 ~ B1*08:01 ~ C*07:01 ~ DRB1*03:01 ~ DQB1*02:01 conferred an increased risk of low humoral response. Considering cellular responses, 50% of the vaccinated subjects responded against Ag1 and 59% against Ag2. Carriers of DRB1*15:01 displayed a higher cellular response both to Ag1 and Ag2 compared to the rest of the cohort. Similarly, DRB1*13:02 predisposed to a robust cellular response to Ag1 and Ag2, while DRB1*11:04 showed an opposite trend. Cellular and humoral responses to Comirnaty are influenced by HLA. Humoral response is mainly associated to class I alleles, with A*03:01, previously associated to protection against severe COVID‐19, and response to vaccination, standing out. Cellular response predominantly involves class II alleles, with DRB1*15:01 and DPB1*13:01 prevailing. Affinity analysis for Spyke peptides is generally in line with the association results. [ABSTRACT FROM AUTHOR]

Background: Methylmalonic aciduria and homocystinuria, CblC type (OMIM #277400) is the most common disorder of cobalamin intracellular metabolism, an autosomal recessive disease, whose biochemical hallmarks are hyperhomocysteinemia, methylmalonic aciduria and low plasma methionine. Despite being a well-recognized disease for pediatricians, there is scarce awareness of its adult presentation. A thorough analysis and discussion of cobalamin C defect presentation in adult patients has never been extensively performed. This article reviews the published data and adds a new case of the latest onset of symptoms ever described for the disease.Results: We present the emblematic case of a 45-year-old male, describing the diagnostic odyssey he ventured through to get to the appropriate treatment and molecular diagnosis. Furthermore, available clinical, biochemical and molecular data from 22 reports on cases and case series were collected, resulting in 45 adult-onset CblC cases, including our own. We describe the onset of the disease in adulthood, encompassing neurological, psychiatric, renal, ophthalmic and thromboembolic symptoms. In all cases treatment with intramuscular hydroxycobalamin was effective in reversing symptoms. From a molecular point of view adult patients are usually compound heterozygous carriers of a truncating and a non-truncating variant in the MMACHC gene.Conclusion: Adult onset CblC disease is a rare disorder whose diagnosis can be delayed due to poor awareness regarding its presenting insidious symptoms and biochemical hallmarks. To avoid misdiagnosis, we suggest that adult onset CblC deficiency is acknowledged as a separate entity from pediatric late onset cases, and that the disease is considered in the differential diagnosis in adult patients with atypical hemolytic uremic syndromes and/or slow unexplained decline in renal function and/or idiopathic neuropathies, spinal cord degenerations, ataxias and/or recurrent thrombosis and/or visual field defects, maculopathy and optic disc atrophy. Plasma homocysteine measurement should be the first line for differential diagnosis when the disease is suspected. To further aid diagnosis, it is important that genes belonging to the intracellular cobalamin pathway are included within gene panels routinely tested for atypical hemolytic uremic syndrome and chronic kidney disorders. [ABSTRACT FROM AUTHOR]

Extracellular adenosine (ADO), generated from ATP or ADP through the concerted action of the ectoenzymes CD39 and CD73, elicits autocrine and paracrine effects mediated by type 1 purinergic receptors. We have tested whether the expres- sion of CD39 and CD73 by chronic lymphocytic leukemia (CLL) cells activates an adenosinergic axis affecting growth and survival. By immunohistochemistry, CD39 is widely expressed in CLL lymph nodes, whereas CD73 is restricted to proliferation centers. CD73 expression is highest on Ki-67- CLL cells, adjacent to T lymphocytes, and is further localized to perivascular areas. CD39+/CD73+ CLL cells generate ADO from ADP in a time- and concentration-dependent manner. In peripheral blood, CD73 expression occurs in 97/299 (32%) CLL patients and pairs with CD38 and ZAP-70 expression. CD73-generated extracellular ADO activates type 1 purinergic A2A receptors that are constitutively expressed by CLL cells and that are further elevated in proliferating neoplastic cells. Activation of the ADO receptors Increases cytoplasmic cAMP levels, inhibiting chemotaxis and limiting spontaneous drug-Induced apoptosis of CLL cells. These data are consistent with the existence of an autocrine adenosinergic loop, and support engraftment of leukemic cells in growth-favorable niches, while simultaneously protecting from the action of chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

CD38 is an ectoenzyme involved in transmembrane signaling and cell adhesion and is used as a disease marker for leukemias and myeloma. CD38 is a dependable negative prognostic marker for chronic lymphocytic leukemia (CLL). Recent evidence indicates that CD38 is a component of a complex network delivering growth and survival signals to CLL cells. In conjunction with chemokines and their receptors, CD38 also influences cell migratory responses. These considerations are the rationale for devising a CLL therapy that uses CD38 as the target. The use of reagents specifically blocking the molecule might provide a new approach for interfering with deleterious growth circuits, therefore increasing the susceptibility of leukemic cells to conventional chemotherapy. [Copyright &y& Elsevier]

Donor-derived cell-free DNA (dd-cfDNA) is considered a reliable marker of organ damage with potential applications in the follow-up of transplant recipients. In this work we present an assay based on the donor-recipient HLA-mismatch (human leukocyte antigen) at the HLA-DRB1 locus to monitor rejection by quantifying the percentage of dd-cfDNA using a droplet digital PCR (polymerase chain reaction) technique. A panel of probes targeting the HLA-DRB1 locus and covering >85% genetic variability was validated and used to assess dd-cfDNA levels in a prospective cohort of 19 adult heart transplant recipients (mean age 50.9±14.8 years). The assay was carried out on a total of 232 liquid biopsies collected at the same time as endomyocardial biopsy (EMB) during routine post-transplant follow-up. Results show a significant increase of dd-cfDNA related to ischemia-reperfusion injury (2.22±2.09%) and to acute cellular rejection (1.71±3.10%) compared to stable conditions (0.43±1.04%, p < 0.0001). On the contrary, no increase was observed during infections or vascular complications, underlining the potential role of this biomarker for rejection monitoring. With a cut-off of 0.11%, the test showed 70.8% specificity (95% CI, 58.17% - 81.40%) and 64.2% sensitivity (95% CI, 49.80% - 76.86%) in discriminating acute rejection from no rejection. These data demonstrate that this HLA mismatch-based droplet digital PCR method is effective for monitoring rejection in heart transplant recipients. Compared to next generation sequencing approaches, it is far more flexible, less expensive and provides faster results. [ABSTRACT FROM AUTHOR]

Aim: Nicotinamide adenine dinucleotide (NAD+) plays central roles in a wide array of normal and pathological conditions. Inhibition of NAD+ biosynthesis can be exploited therapeutically in cancer, including melanoma. To obtain quantitation of NAD+ levels in live cells and to address the issue of the compartmentalization of NAD+ biosynthesis, we exploited a recently described genetically encoded NAD+ biosensor (LigA-circularly permutated Venus), which was targeted to the cytosol, mitochondria, and nuclei of BRAF-V600E A375 melanoma cells, a model of metastatic melanoma (MM). Results: FK866, a specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), the main NAD+-producing enzyme in MM cells, was used to monitor NAD+ depletion kinetics at the subcellular level in biosensor-transduced A375 cells. In addition, we treated FK866-blocked A375 cells with NAD+ precursors, including nicotinamide, nicotinic acid, nicotinamide riboside, and quinolinic acid, highlighting an organelle-specific capacity of each substrate to rescue from NAMPT block. Expression of NAD+ biosynthetic enzymes was then biochemically studied in isolated organelles, revealing the presence of NAMPT in all three cellular compartments, whereas nicotinate phosphoribosyltransferase was predominantly cytosolic and mitochondrial, and nicotinamide riboside kinase mitochondrial and nuclear. In keeping with biosensor data, quinolinate phosphoribosyltransferase was expressed at extremely low levels. Innovation and Conclusions: Throughout this work, we validated the use of genetically encoded NAD+ biosensors to characterize subcellular distribution of NAD+ production routes in MM. The chance of real-time monitoring of NAD+ fluctuations after chemical perturbations, together with a deeper comprehension of the cofactor biosynthesis compartmentalization, strengthens the foundation for a targeted strategy of NAD+ pool manipulation in cancer and metabolic diseases. [ABSTRACT FROM AUTHOR]

Background: One of the effects of oncogenic signaling is metabolic reprogramming of tumor cells to support anabolic growth, opening the way to therapeutic targeting of metabolic pathways.Methods: We studied NAD biosynthesis in BRAF inhibitor (BRAFi)-resistant (BiR) melanoma cell lines. Data in cell lines were confirmed by immunohistochemistry in biopsies from 17 patients with metastatic melanoma (MM) before and after the acquisition of resistance to BRAFi. Therapeutic potential of NAD biosynthesis inhibitors was determined by invitro monitoring cell growth and death and in mouse xenograft models. Mice (n = 6-10 mice/group) were treated with nicotinamide phosphoribosyltranferase inhibitor (NAMPTi), BRAFi, or their combination, and tumor growth and survival were analyzed. All statistical tests were two-sided.Results: BiR cells had higher NAD levels compared with their BRAFi-sensitive counterparts (P < .001 and P = .001 for M14 and A375, respectively) and with normal melanocytes (P < .001), achieved through transcriptional upregulation of the enzyme NAMPT, which became the master regulator of NAD synthesis. Conversely, treatment with BRAFi or MEK inhibitors decreased NAMPT expression and cellular NAD levels. Robust NAMPT upregulation was documented in tissue biopsies from MM patients after development of resistance to BRAFi (P < .001). Treatment of melanoma cells with NAMPTi depleted NAD and ATP, depolarized mitochondrial membrane, and led to reactive oxygen species production, blocking cells in the G2/M phase and inducing apoptosis. Treatment of BiR xenografts with NAMPTi improved mouse survival (median survival of vehicle-treated mice was 52 days vs 100 days for NAMPTi-treated ones in M14/BiR, while in A375/BiR median survival of vehicle-treated mice was 23.5 days vs 43 days for NAMPTi-treated ones, P < .001).Conclusions: BiR melanoma cells overexpress NAMPT, which acts as a connecting element between BRAF oncogenic signaling and metabolism, becoming an actionable target for this subset of MM patients. [ABSTRACT FROM AUTHOR]

Chronic lymphocytic leukemia (CLL) is a variable disease; therefore, markers to identify aggressive forms are essential for patient management. Here, we have shown that expression of the costimulatory molecule and microbial sensor SLAMF1 (also known as CD150) is lost in a subset of patients with an aggressive CLL that associates with a shorter time to first treatment and reduced overall survival. SLAMF1 silencing in CLL-like Mec-1 cells, which constitutively express SLAMF1, modulated pathways related to cell migration, cytoskeletal organization, and intracellular vesicle formation and recirculation. SLAMF1 deficiency associated with increased expression of CXCR4, CD38, and CD44, thereby positively affecting chemotactic responses to CXCL12. SLAMF1 ligation with an agonistic monoclonal antibody increased ROS accumulation and induced phosphorylation of p38, JNK1/2, and BCL2, thereby promoting the autophagic flux. Beclin1 dissociated from BCL2 in response to SLAMF1 ligation, resulting in formation of the autophagy macrocomplex, which contains SLAMF1, beclin1, and the enzyme VPS34. Accordingly, SLAMF1-silenced cells or SLAMF1(lo) primary CLL cells were resistant to autophagy-activating therapeutic agents, such as fludarabine and the BCL2 homology domain 3 mimetic ABT-737. Together, these results indicate that loss of SLAMF1 expression in CLL modulates genetic pathways that regulate chemotaxis and autophagy and that potentially affect drug responses, and suggest that these effects underlie unfavorable clinical outcome experienced by SLAMF1(lo) patients. [ABSTRACT FROM AUTHOR]

The Human Genome Project in 2001 has opened the Pandora's box on the complexity of DNA structure and transcriptional regulation. Only a small fraction of the 3 billion bases is part of the protein-coding genes, while approximately 98.5% is represented by non-coding sequences. Besides the classical messenger, ribosomal and transfer RNAs, the "cellular RNA world" is made of short and long non-coding RNAs (lncRNAs) that play regulatory or structural roles, shifting the balance of pathogenic gene variations from coding to non-coding genome. LncRNAs are 200 and 100,000 nucleotide long molecules, not translated into protein, highly heterogeneous in terms of expression within the cells, showing tissue and stage specificity. They are emerging as modifiers of epigenetic, transcription, and translation processes, and can be implicated in the pathogenesis of cancers. In this review, we will focus on B, T and NK hematological malignancies, with the aim of presenting an update on lncRNAs landscape and on their role as potential oncogenes or oncosuppressors. Moreover, we will talk over the bi-directional crosstalk between lncRNAs and epigenetics since these modifications can impact on lncRNAs expression, and, in turn, non-coding transcripts can regulate chromatin organization and transcriptional processes. Finally, we will point the attention on their use as potential biomarkers for diagnostic and prognostic purposes, and possibly as attractive targets in a translational perspective, opening for novel therapeutic options. [ABSTRACT FROM AUTHOR]

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis. In the extracellular compartment, it exhibits cytokine-/adipokinelike properties, suggesting that it stands at the crossroad between metabolism and inflammation. Here we show that both intracellular and extracellular NAMPT levels are increased in cells and plasma of chronic lymphocytic leukemia (CLL) patients. The extracellular form (eNAMPT) is produced by CLL lymphocytes upon B-cell receptor, Toll-like receptor, and nuclear factor κB (NF-κB) signaling pathway activation. eNAMPT is important for differentiation of resting monocytes, polarizing them toward tumor-supporting M2 macrophages. These cells express high levels of CD163, CD206, and indoleamine 2,3-dioxygenase and secrete immunosuppressive (interleukin [IL] 10, CC chemokine ligand 18) and tumor-promoting (IL-6, IL-8) cytokines. NAMPT-primed M2 macrophages activate extracellular-regulated kinase 1/2, signal transducer and activator of transcription 3, and NF-κB signaling; promote leukemic cell survival; and reduce T-cell responses. These effects are independent of the enzymatic activity of NAMPT, as inferred from the use of an enzymatically inactive mutant. Overall, these results reveal that eNAMPT is a critical element in the induction of an immunosuppressive and tumor-promoting microenvironment of CLL. [ABSTRACT FROM AUTHOR]

3MC syndrome is an autosomal recessive disorder encompassing four rare disorders previously known as the Malpuech, Michels, Mingarelli and Carnevale syndromes. They are characterized by a variable spectrum of abnormalities, including facial dysmorphisms, along with genital, limb and vesico-renal anomalies. The syndrome was originally attributed to mutations in MASP1 and COLEC11 , which code for proteins involved in the lectin complement pathway. More recently, mutations in COLEC10 , a third gene coding for collectin CL-L1, were identified in a limited number of patients with 3MC syndrome. Here we describe a 4-years-old patient with typical 3MC phenotypic characteristics, including blepharophimosis, telecanthus, high arched eyebrows, fifth finger clinodactyly, sacral dimple and horseshoe kidney. Initial genetic analysis was based on clinical exome sequencing, where only MASP1 and COLEC11 genes are present, without evidence of pathogenic variants. Sanger sequencing of COLEC10 identified the homozygous frameshift variant c.807_810delCTGT; p.Cys270Serfs*33, which results in the loss of the natural stop codon. The resulting protein is 24 amino acids longer and lacks a conserved cysteine residue (Cys270), which could affect protein folding. Segregation studies confirmed that both parents were carriers for the variant: interestingly they originate from the same area of Apulia in southern Italy. Plasma levels of CL-L1 in the patient and her parents were within normal range, suggesting that this variant does not modify transcription or secretion. However, the variant affects the chemo-attractive feature of CL-L1, as HeLa cells migrate significantly less in response to the mutant protein compared to the wild-type one. [ABSTRACT FROM AUTHOR]

The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications such as chemorefractoriness. In the present study, BIRC3, a negative regulator of noncanonical NF-&κgr;Β signaling, was investigated in different CLL clinical phases. BIRC3 lesions were absent in monoclonal B-celi lymphocytosis (0 of 63) and were rare in CLL at diagnosis (13 of 306, 4%). Conversely, BIRC3 disruption selectively affected 12 of 49 (24%) fludarabine-refractory CLL cases by inactivating mutations and/or gene deletions that distributed in a mutually exclusive fashion with TP53abnormalities. In contrast to fludarabinerefractory CLL, progressive but fludarabine-sensitive patients were consistently devoid of BIRC3 abnormalities, suggesting that BIRC3 genetic lesions associate specifically with a chemorefractory phenotype. By actuarial analysis in newly diagnosed CLL (η = 306), BIRC3 disruption identified patients with a poor outcome similar to that associated with TP53 abnormalities and exerted a prognostic role that was independent of widely accepted clinical and genetic risk factors. Consistent with the role of BIRC3 as a negative regulator of NF-&κgr;Β, biochemical studies revealed the presence of constitutive noncanonical NF-&κgr;Β activation in fludarabinerefractory CLL patients harboring molecular lesions of BIRC3. These data identify BIRC3 disruption as a recurrent genetic lesion of high-riskCLL devoid of TP53abnormalities. [ABSTRACT FROM AUTHOR]

The genetic lesions identified in chronic lymphocytic leukemia (CLL) do not entirely recapitulate the disease pathogenesis and the development of serious complications, such as chemorefractoriness. While investigating the coding genome of fludarabine-refractory CLL, we observed that mutations of SF3B1, encoding a splicing factor and representing a critical component of the cell spliceosome, were recurrent in 10 of 59 (17%) fludarabine-refractory cases, with a frequency significantly greater than that observed in a consecutive CLL cohort sampled at diagnosis (17/301, 5%; P = .002). Mutations were somatically acquired, were generally represented by missense nucleotide changes, clustered in selected HEAT repeats of the SF3B1 protein, recurrently targeted 3 hotspots (codons 662, 666, and 700), and were predictive of a poor prognosis. In fludarabine-refractory CLL, SF3B1 mutations and TP53 disruption distributed in a mutually exclusive fashion (P = .046). The identification of SF3B1 mutations points to splicing regulation as a novel pathogenetic mechanism of potential clinical relevance in CLL. [ABSTRACT FROM AUTHOR]

Splenic marginal zone lymphoma (SMZL) is one of the few B-cell lymphoma types that remain orphan of molecular lesions in cancer-related genes. Detection of active NF-κB signaling in 14 (58%) of 24 SMZLs prompted the investigation of NF-κB molecular alterations in 101 SMZLs. Mutations and copy number abnormalities of NF-κB genes occurred in 36 (36%) of 101 SMZLs and targeted both canonical (TNFAIP3 and IKBKB) and noncanonical (BIRC3, TRAF3, MAP3K14) NF-κB pathways. Most alterations were mutually exclusive, documenting the existence of multiple independent mechanisms affecting NF-κB in SMZL. BIRC3 inactivation in SMZL recurred because of somatic mutations that disrupted the same RING domain that in extranodal marginal zone lymphoma is removed by the t(11;18) translocation, which points to BIRC3 disruption as a common mechanism across marginal zone B-cell lymphomagenesis. Genetic lesions of NF-κB provide a molecular basis for the pathogenesis of more than 30% of SMZLs and offer a suitable target for NF-κB therapeutic approaches in this lymphoma. [ABSTRACT FROM AUTHOR]

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukaemia (CLL) to aggressive lymphoma. Risk factors of CLL transformation to RS are only partly known. We explored the role of the host genetic background as a risk factor for RS occurrence. Forty-five single nucleotide polimorphisms (SNPs) known to be relevant for CLL prognosis were genotyped in a consecutive cohort of 331 CLL, of which 21 had transformed to RS. After correcting for multiple testing and adjusting for previously reported RS risk factors, the LRP4 rs2306029 TT variant genotype was the sole SNP independently associated with a higher risk of RS transformation (Hazard Ratio: 4·17; P = 0·001; q = 0·047). The enrichment of LRP4 TT genotype in RS was confirmed in an independent series ( n = 44) used for validation purposes. The LRP4 protein was expressed in CLL ( n = 66). Bioinformatic analysis scored LRP4 rs2306029 as a variant with possible deleterious and damaging variant of LRP4. LRP4 genotyping may help the recognition of patients with increased risk of RS at the time of CLL diagnosis. [ABSTRACT FROM AUTHOR]

Human CD38 is a pleiotropic glycoprotein belonging to a family of enzymes/receptors involved in the catabolism of extracellular nucleotides. CD38-receptor activities are regulated through binding to the nonsubstrate ligand CD31. CD38 expression above a critical threshold is a negative prognostic marker for chronic lymphocytic leukemia (CLL) patients. Activation of CD38 by means of agonistic monoclonal antibodies or the CD31 ligand induces proliferation and immunoblast differentiation of CLL cells. Here we define the genetic signature that follows long-term in vitro interactions between CD38+ CLL lymphocytes and CD31+ cells. The emerging profile confirms that the CD31/CD38 axis activates genetic programs relevant for proliferative responses. It also indicates a contribution of this pathway to the processes mediating migration and homing. These results further support the notion that the CD31/CD38 axis is part of a network of accessory signals that modify the microenvironment, favoring localization of leukemic cells to growth-permissive sites. [ABSTRACT FROM AUTHOR]

This paper reviews some of the results and the speculations presented at the Torino CD38 Meeting in June, 2006 and focused on CD38 and CD157 seen as a family of molecules acting as surface receptors of immune cells. This partisan view was adopted in the attempt to combine the enzymatic functions with what the immunologists consider key functions in different cell models. At the moment, it is unclear whether the two functions are correlated, indifferent, or independent. Here we present conclusions inferred exclusively on human cell models, namely T and B lymphocytes, dendritic cells, and granulocytes. As an extra analytical tool, we try to follow in the history of life when the enzymatic and receptorial functions were generated, mixing ontogeny, membrane localization, and cell anchorage. [ABSTRACT FROM AUTHOR]

Simple Summary: Malignant melanoma (MM) is the most fatal skin cancer due to its high metastatic potential. Treatment strategies are dramatically changing due to the introduction of BRAF/MEK inhibitors (i) and immunotherapy; however, multiple resistant mechanisms rapidly occur including metabolic rewiring. This study aimed to establish the driver role of the nicotinamide adenine dinucleotide (NAD)-biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in BRAFi resistance development. We defined that NAMPT over-expressing MM cells were strikingly similar to cells that acquired resistance to BRAFi in terms of growth, invasion, and phenotype plasticity. These findings confirmed NAMPT as a key factor in melanoma progression and in the onset of BRAFi resistance in melanoma patients, opening new therapeutic possibilities for this subset of patients. Serine–threonine protein kinase B-RAF (BRAF)-mutated metastatic melanoma (MM) is a highly aggressive type of skin cancer. Treatment of MM patients using BRAF/MEK inhibitors (BRAFi/MEKi) eventually leads to drug resistance, limiting any clinical benefit. Herein, we demonstrated that the nicotinamide adenine dinucleotide (NAD)-biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) is a driving factor in BRAFi resistance development. Using stable and inducible NAMPT over-expression systems, we showed that forced NAMPT expression in MM BRAF-mutated cell lines led to increased energy production, MAPK activation, colony-formation capacity, and enhance tumorigenicity in vivo. Moreover, NAMPT over-expressing cells switched toward an invasive/mesenchymal phenotype, up-regulating expression of ZEB1 and TWIST, two transcription factors driving the epithelial to mesenchymal transition (EMT) process. Consistently, within the NAMPT-overexpressing cell line variants, we observed an increased percentage of a rare, drug-effluxing stem cell-like side population (SP) of cells, paralleled by up-regulation of ABCC1/MRP1 expression and CD133-positive cells. The direct correlation between NAMPT expression and gene set enrichments involving metastasis, invasiveness and mesenchymal/stemness properties were verified also in melanoma patients by analyzing The Cancer Genome Atlas (TCGA) datasets. On the other hand, CRISPR/Cas9 full knock-out NAMPT BRAFi-resistant MM cells are not viable, while inducible partial silencing drastically reduces tumor growth and aggressiveness. Overall, this work revealed that NAMPT over-expression is both necessary and sufficient to recapitulate the BRAFi-resistant phenotype plasticity. [ABSTRACT FROM AUTHOR]

Representing the major cause of morbidity and mortality for chronic lymphocytic leukemia (CLL) patients, immunosuppression is a common feature of the disease. Effectors of the innate and the adaptive immune response show marked dysfunction and skewing towards the generation of a tolerant environment that favors disease expansion. Major deregulations are found in the T lymphocyte compartment, with inhibition of CD8+ cytotoxic and CD4+ activated effector T cells, replaced by exhausted and more tolerogenic subsets. Likewise, differentiation of monocytes towards a suppressive M2-like phenotype is induced at the expense of pro-inflammatory sub-populations. Thanks to their B-regulatory phenotype, leukemic cells play a central role in driving immunosuppression, progressively inhibiting immune responses. A number of signaling cascades triggered by soluble mediators and cell–cell contacts contribute to immunomodulation in CLL, fostered also by local environmental conditions, such as hypoxia and derived metabolic acidosis. Specifically, molecular pathways modulating T-cell activity in CLL, spanning from the best known cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death 1 (PD-1) to the emerging T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT)/CD155 axes, are attracting increasing research interest and therapeutic relevance also in the CLL field. On the other hand, in the microenvironment, the B cell receptor (BCR), which is undoubtedly the master regulator of leukemic cell behavior, plays an important role in orchestrating immune responses, as well. Lastly, local conditions of hypoxia, typical of the lymphoid niche, have major effects both on CLL cells and on non-leukemic immune cells, partly mediated through adenosine signaling, for which novel specific inhibitors are currently under development. In summary, this review will provide an overview of the molecular and microenvironmental mechanisms that modify innate and adaptive immune responses of CLL patients, focusing attention on those that may have therapeutic implications. [ABSTRACT FROM AUTHOR]