Genome editing and cancer therapy: handling the hypoxia-responsive pathway as a promising strategy.

The precise characterization of oxygen-sensing pathways and the identification of pO2-regulated gene expression are both issues of critical importance. The O2-sensing system plays crucial roles in almost all the pivotal human processes, including the stem cell specification, the growth and development of tissues (such as embryogenesis), the modulation of intermediate metabolism (including the shift of the glucose metabolism from oxidative to anaerobic ATP production and vice versa), and the control of blood pressure. The solid cancer microenvironment is characterized by low oxygen levels and by the consequent activation of the hypoxia response that, in turn, allows a complex adaptive response characterized mainly by neoangiogenesis and metabolic reprogramming. Recently, incredible advances in molecular genetic methodologies allowed the genome editing with high efficiency and, above all, the precise identification of target cells/tissues. These new possibilities and the knowledge of the mechanisms of adaptation to hypoxia suggest the effective development of new therapeutic approaches based on the manipulation, targeting, and exploitation of the oxygen-sensor system molecular mechanisms.

Bone Health Impairment in Patients with Hemoglobinopathies: From Biological Bases to New Possible Therapeutic Strategies.

Hemoglobinopathies are monogenic disorders affecting hemoglobin synthesis. Thalassemia and sickle cell disease (SCD) are considered the two major hemoglobinopathies. Thalassemia is a genetic disorder and one of the major hemoglobinopathies determined by an impairment of globin chain production, which causes an alteration of erythropoiesis, an improvement in hemolysis, and an alteration of iron homoeostasis. In SCD, the mutations are on the ß-globin chain of hemoglobin which results in a substitution of glutamic acid by valine with consequent formation of Hemoglobin S (HbS). Several factors are involved in bone metabolism alteration in patients with hemoglobinopathies, among them hormonal deficiency, bone marrow hyperplasia, iron overload, inflammation, and increased bone turnover. Bone metabolism is the result of balance maintenance between bone deposition and bone resorption, by osteoblasts (OBs) and osteoclasts (OCs). An impairment of this balance is responsible for the onset of bone diseases, such as osteoporosis (OP). Therefore, here we will discuss the alteration of bone metabolism in patients with hemoglobinopathies and the possible therapeutic strategies to contain and/or counteract bone health impairment in these patients, taking into consideration not only the pharmacological treatments already used in the clinical armamentarium, but also the new possible therapeutic strategies.

Effects of Germline VHL Deficiency on Growth, Metabolism, and Mitochondria.

Mutations in VHL, which encodes von Hippel-Lindau tumor suppressor (VHL), are associated with divergent diseases. We describe a patient with marked erythrocytosis and prominent mitochondrial alterations associated with a severe germline VHL deficiency due to homozygosity for a novel synonymous mutation (c.222C→A, p.V74V). The condition is characterized by early systemic onset and differs from Chuvash polycythemia (c.598C→T) in that it is associated with a strongly reduced growth rate, persistent hypoglycemia, and limited exercise capacity. We report changes in gene expression that reprogram carbohydrate and lipid metabolism, impair muscle mitochondrial respiratory function, and uncouple oxygen consumption from ATP production. Moreover, we identified unusual intermitochondrial connecting ducts. Our findings add unexpected information on the importance of the VHL-hypoxia-inducible factor (HIF) axis to human phenotypes. (Funded by Associazione Italiana Ricerca sul Cancro and others.).

CB2 Receptor as Emerging Anti-Inflammatory Target in Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a very severe X-linked dystrophinopathy. It is due to a mutation in the DMD gene and causes muscular degeneration in conjunction with several secondary co-morbidities, such cardiomyopathy and respiratory failure. DMD is characterized by a chronic inflammatory state, and corticosteroids represent the main therapy for these patients. To contradict drug-related side effects, there is need for novel and more safe therapeutic strategies. Macrophages are immune cells stringently involved in both physiological and pathological inflammatory processes. They express the CB2 receptor, one of the main elements of the endocannabinoid system, and have been proposed as an anti-inflammatory target in several inflammatory and immune diseases. We observed a lower expression of the CB2 receptor in DMD-associated macrophages, hypothesizing its involvement in the pathogenesis of this pathology. Therefore, we analyzed the effect of JWH-133, a CB2 receptor selective agonist, on DMD-associated primary macrophages. Our study describes the beneficial effect of JWH-133 in counteracting inflammation by inhibiting pro-inflammatory cytokines release and by directing macrophages' phenotype toward the M2 anti-inflammatory one.

Thalassaemia is paradoxically associated with a reduced risk of in-hospital complications and mortality in COVID-19: Data from an international registry.

Although numerous patient-specific co-factors have been shown to be associated with worse outcomes in COVID-19, the prognostic value of thalassaemic syndromes in COVID-19 patients remains poorly understood. We studied the outcomes of 137 COVID-19 patients with a history of transfusion-dependent thalassaemia (TDT) and transfusion independent thalassaemia (TIT) extracted from a large international cohort and compared them with the outcomes from a matched cohort of COVID-19 patients with no history of thalassaemia. The mean age of thalassaemia patients included in our study was 41 ± 16 years (48.9% male). Almost 81% of these patients suffered from TDT requiring blood transfusions on a regular basis. 38.7% of patients were blood group O. Cardiac iron overload was documented in 6.8% of study patients, whereas liver iron overload was documented in 35% of study patients. 40% of thalassaemia patients had a history of splenectomy. 27.7% of study patients required hospitalization due to COVID-19 infection. Amongst the hospitalized patients, one patient died (0.7%) and one patient required intubation. Continuous positive airway pressure (CPAP) was required in almost 5% of study patients. After adjustment for age-, sex- and other known risk factors (cardiac disease, kidney disease and pulmonary disease), the rate of in-hospital complications (supplemental oxygen use, admission to an intensive care unit for CPAP therapy or intubation) and all-cause mortality was significantly lower in the thalassaemia group compared to the matched cohort with no history of thalassaemia. Amongst thalassaemia patients in general, the TIT group exhibited a higher rate of hospitalization compared to the TDT group (p = 0.001). In addition, the rate of complications such as acute kidney injury and need for supplemental oxygen was significantly higher in the TIT group compared to the TDT group. In the multivariable logistic regression analysis, age and history of heart or kidney disease were all found to be independent risk factors for increased in-hospital, all-cause mortality, whereas the presence of thalassaemia (either TDT or TIT) was found to be independently associated with reduced all-cause mortality. The presence of thalassaemia in COVID-19 patients was independently associated with lower in-hospital, all-cause mortality and few in-hospital complications in our study. The pathophysiology of this is unclear and needs to be studied in vitro and in animal models.

Risk factors for endocrine complications in transfusion-dependent thalassemia patients on chelation therapy with deferasirox: a risk assessment study from a multi-center nation-wide cohort.

Transfusion-dependent patients typically develop iron-induced cardiomyopathy, liver disease, and endocrine complications. We aimed to estimate the incidence of endocrine disorders in transfusiondependent thalassemia (TDT) patients during long-term iron-chelation therapy with deferasirox (DFX). We developed a multi-center follow-up study of 426 TDT patients treated with once-daily DFX for a median duration of 8 years, up to 18.5 years. At baseline, 118, 121, and 187 patients had 0, 1, or ≥2 endocrine diseases respectively. 104 additional endocrine diseases were developed during the follow-up. The overall risk of developing a new endocrine complication within 5 years was 9.7% (95% Confidence Interval [CI]: 6.3-13.1). Multiple Cox regression analysis identified three key predictors: age showed a positive log-linear effect (adjusted hazard ratio [HR] for 50% increase 1.2, 95% CI: 1.1-1.3, P=0.005), the serum concentration of thyrotropin showed a positive linear effect (adjusted HR for 1 mIU/L increase 1.3, 95% CI: 1.1-1.4, P<0.001) regardless the kind of disease incident, while the number of previous endocrine diseases showed a negative linear effect: the higher the number of diseases at baseline the lower the chance of developing further diseasess (adjusted HR for unit increase 0.5, 95% CI: 0.4-0.7, P<0.001). Age and thyrotropin had similar effect sizes across the categories of baseline diseases. The administration of levothyroxine as a covariate did not change the estimates. Although in DFX-treated TDT patients the risk of developing an endocrine complication is generally lower than the previously reported risk, there is considerable risk variation and the burden of these complications remains high. We developed a simple risk score chart enabling clinicians to estimate their patients' risk. Future research will look at increasing the amount of variation explained from our model and testing further clinical and laboratory predictors, including the assessment of direct endocrine magnetic resonance imaging.

Effect of CB2 Stimulation on Gene Expression in Pediatric B-Acute Lymphoblastic Leukemia: New Possible Targets.

Acute lymphoblastic leukemia type B (B-ALL) is the most common kind of pediatric leukemia, characterized by the clonal proliferation of type B lymphoid stem cells. Important progress in ALL treatments led to improvements in long-term survival; nevertheless, many adverse long-term consequences still concern the medical community. Molecular and cellular target therapies, together with immunotherapy, are promising strategies to overcome these concerns. Cannabinoids, enzymes involved in their metabolism, and cannabinoid receptors type 1 (CB1) and type 2 (CB2) constitute the endocannabinoid system, involved in inflammation, immune response, and cancer. CB2 receptor stimulation exerts anti-proliferative and anti-invasive effects in many tumors. In this study, we evaluated the effects of CB2 stimulation on B-ALL cell lines, SUP-B15, by RNA sequencing, Western blotting, and ELISA. We observe a lower expression of CB2 in SUP-B15 cells compared to lymphocytes from healthy subjects, hypothesizing its involvement in B-ALL pathogenesis. CB2 stimulation reduces the expression of CD9, SEC61G, TBX21, and TMSB4X genes involved in tumor growth and progression, and also negatively affects downstream intracellular pathways. Our findings suggest an antitumor role of CB2 stimulation in B-ALL, and highlight a functional correlation between CB2 receptors and specific anti-tumoral pathways, even though further investigations are needed.

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality.

Band 3 (anion exchanger 1; AE1) is the most abundant membrane protein in red blood cells, which in turn are the most abundant cells in the human body. A compelling model posits that, at high oxygen saturation, the N-terminal cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during red blood cell aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of the quality of stored blood and efficacy of transfusion, a life-saving intervention for millions of recipients worldwide. Here we leveraged two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of the role of this protein in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and crosslinking proteomics, we provide a map of the red blood cell interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase. As a proof-of-principle and to provide further mechanistic evidence of the role of AE1 in the regulation of redox homeo stasis of stored red blood cells, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recovery of stored red blood cells from healthy human donors and genetically ablated mice.

Headache in beta-thalassemia: An Italian multicenter clinical, conventional MRI and MR-angiography case-control study.

OBJECTIVES: A strikingly increased headache prevalence was recently noted in Sri Lankan beta-thalassemia patients, raising several concerns regarding long-term neurological involvement in this condition. METHODS: We interviewed on headache occurrence and characteristics 102 Italian beta-thalassemia patients and 129 healthy controls. 3T-MRI, MR-angiography, MR-venography, cognitive and psychiatric findings were considered. RESULTS: Headache was diagnosed in 39/102 (38.2%) beta-thalassemia patients without significant phenotype-related differences and in 51/129 (39.5%) controls. Patients and controls did not differ significantly regarding episode number (5.9 ± 6.2 vs 5.4 ± 4.4 days/month), subjective severity-score (6.8 ± 1.4 vs 7.1 ± 1.3), age-at-onset (24.3 ± 13.0 vs 19.5 ± 9.6 years) and headache-subtype rate. No main demographic, clinical or laboratory data was associated with headache but female gender. Headache was not associated with white matter lesions (number or maximal diameter), intracranial aneurysms, intracranial artery stenoses or venous sinus thrombosis. Cognitive and psychiatric evaluations were worse in beta-thalassemia, however, headache did not correlate with full-scale Intelligence Quotient (75.4 ± 18.0 vs 76.7 ± 15.3, with and without headache, respectively) or Brief Psychiatric Rating Scale scores (29.1 ± 2.7 vs 28.5 ± 3.4). CONCLUSIONS: Among Italian beta-thalassemia patients, headache does not seem to be more common or severe than in the general population. In addition, patients with headache do not seem to present increased conventional MRI, MR-angiography and cognitive/psychiatric changes.

Effects of Eltrombopag on In Vitro Macrophage Polarization in Pediatric Immune Thrombocytopenia.

Immune Thrombocytopenia (ITP) is an autoimmune disease characterized by autoantibodies-mediated platelet destruction, a prevalence of M1 pro-inflammatory macrophage phenotype and an elevated T helper 1 and T helper 2 lymphocytes (Th1/Th2) ratio, resulting in impairment of inflammatory profile and immune response. Macrophages are immune cells, present as pro-inflammatory classically activated macrophages (M1) or as anti-inflammatory alternatively activated macrophages (M2). They have a key role in ITP, acting both as effector cells, phagocytizing platelets, and, as antigen presenting cells, stimulating auto-antibodies against platelets production. Eltrombopag (ELT) is a thrombopoietin receptor agonist licensed for chronic ITP to stimulate platelet production. Moreover, it improves T and B regulatory cells functions, suppresses T-cells activity, and inhibits monocytes activation. We analyzed the effect of ELT on macrophage phenotype polarization, proposing a new possible mechanism of action. We suggest it as a mediator of macrophage phenotype switch from the M1 pro-inflammatory type to the M2 anti-inflammatory one in paediatric patients with ITP, in order to reduce inflammatory state and restore the immune system function. Our results provide new insights into the therapy and the management of ITP, suggesting ELT also as immune-modulating drug.

No increased cerebrovascular involvement in adult beta-thalassemia by advanced MRI analyses.

Beta-thalassemia-related anemia and chronic hypercoagulative state are supposed to cause cumulative cerebrovascular damage with consequent parenchymal/vascular changes and functional impairment. However, recent conventional MRI/MR-angiography investigations failed to show an increased cerebrovascular involvement in beta-thalassemia patients managed according to current treatment guidelines, in spite of significantly decreased full-scale IQ scores. We therefore investigated those patients and controls by means of advanced quantitative MRI analyses (based on magnetization transfer and diffusion tensor imaging) searching for signs of possible cerebrovascular injuries undetected by conventional MRI/MR-angiography. The 3 T-MRI study protocol included diffusion tensor imaging and 3D-multi-echo FLASH sequences for magnetization transfer analysis. Whole-brain voxel-based analyses showed that magnetization transfer, fractional anisotropy, and mean, radial and axial diffusivity do not differ between healthy controls and beta-thalassemia patients (considered as a whole group or as distinct transfusion dependent and non-transfusion dependent subgroups). No correlation emerged between all the considered MRI metrics and cognitive findings (full-scale IQ) or the main clinical and laboratory data. According to our findings, adult neurologically-asymptomatic beta-thalassemia patients (regardless of clinical severity) do not seem to present an increased disease-related cerebrovascular vulnerability compared to healthy controls downsizing the need of regular brain MRI monitoring, at least when the current treatment guidelines are followed.

Long-term improvement in cardiac magnetic resonance in ß-thalassemia major patients treated with deferasirox extends to patients with abnormal baseline cardiac function.

The management of iron overload in thalassemia has changed dramatically since the implementation of magnetic resonance imaging, which allows detection of preclinical iron overload and prevention of clinical complications. This study evaluated the effect of deferasirox (DFX), the newest once-daily oral chelator, on cardiac function, iron overload and cardiovascular events over a longer follow up in a "real world" setting. Longitudinal changes in cardiac magnetic resonance T2*, cardiac function parameters and cardiovascular clinical events were assessed in a cohort of 98 TM patients exposed to DFX for a mean of 6.9 years (range 1.8-11.6 years). No cardiac death or incident heart failure occurred. Cardiac T2* significantly increased (+2.6 ± 11.9 msec; P = 0.035) in the whole population, with a significantly greater increase (+11.6 ± 15.5 msec, P = 0.019) in patients with cardiac iron overload (T2* <20 ms). A significant improvement in left-ventricular ejection fraction (LVEF) (from 50.6 ± 6 to 60.2 ± 5; P = 0.001) was observed in 11 (84.6%) out of 13 patients who normalized cardiac function (LVEF >56%). Arrhythmias were the most frequent cardiac adverse event noted but none led to DFX discontinuation. Our data indicate that DFX is effective in maintaining cardiac iron level in the normal range and in improving cardiac iron overload. No heart failure or cardiac death was reported over this longer observation up to 12 years. For the first time, a DFX-induced improvement in LVEF was observed in a subgroup of patients with abnormal cardiac function at baseline, a preliminary observation which deserves further evaluation.

Brain perfusion changes in beta-thalassemia.

BACKGROUND: Brain injury in hereditary hemoglobinopathies is commonly attributed to anemia-related relative hypoperfusion in terms of impaired oxygen blood supply. Supratentorial and infratentorial vascular watershed regions seem to be especially vulnerable, but data are very scarce. AIMS: We investigated a large beta-thalassemia sample with arterial spin labeling in order to characterize regional perfusion changes and their correlation with phenotype and anemia severity. METHODS: We performed a multicenter single-scanner cross-sectional 3T-MRI study analyzing non-invasively the brain perfusion in 54 transfusion-dependent thalassemia (TDT), 23 non-transfusion-dependent thalassemia (NTDT) patients and 56 Healthy Controls (HC). Age, hemoglobin levels, and cognitive functioning were recorded. RESULTS: Both TDT and NTDT patients showed globally increased brain perfusion values compared to healthy controls, while no difference was found between patient subgroups. Using age and sex as covariates and scaling the perfusion maps for the global cerebral blood flow, beta-thalassemia patients showed relative hyperperfusion in supratentorial/infratentorial watershed regions. Perfusion changes correlated with hemoglobin levels (p = 0.013) and were not observed in the less severely anemic patients (hemoglobin level > 9.5 g/dL). In the hyperperfused regions, white matter density was significantly decreased (p = 0.0003) in both patient subgroups vs. HC. In NTDT, white matter density changes correlated inversely with full-scale Intelligence Quotient (p = 0.007) while in TDT no correlation was found. CONCLUSION: Relative hyperperfusion of watershed territories represents a hemodynamic hallmark of beta-thalassemia anemia challenging previous hypotheses of brain injury in hereditary anemias. A careful management of anemia severity might be crucial for preventing structural white matter changes and subsequent long-term cognitive impairment.

Role of Nutraceuticals in Counteracting Inflammation in In Vitro Macrophages Obtained from Childhood Cancer Survivors.

The advancement of anti-cancer therapies has markedly improved the survival rate of children with cancer, making them long-term childhood cancer survivors (CCS). Nevertheless, these treatments cause a low-grade inflammatory state, determining inflamm-aging and, thus, favoring the early onset of chronic diseases normally associated with old age. Identification of novel and safer therapeutic strategies is needed to counteract and prevent inflamm-aging. Macrophages are cells involved in immune and inflammatory responses, with a pivotal role in iron metabolism, which is related to inflammation. We obtained macrophages from CCS patients and evaluated their phenotype markers, inflammatory states, and iron metabolism by Western blotting, ELISA, and iron assays. We observed a strong increase in classically activated phenotype markers (M1) and iron metabolism alteration in CCS, with an increase in intracellular iron concentration and inflammatory markers. These results suggest that the prevalence of M1 macrophages and alteration of iron metabolism could be involved in the worsening of inflammation in CCS. Therefore, we propose macrophages and iron metabolism as novel therapeutic targets to counteract inflamm-aging. To avoid toxic regimens, we tested some nutraceuticals (resveratrol, curcumin, and oil-enriched lycopene), which are already known to exert anti-inflammatory properties. After their administration, we observed a macrophage switch towards the anti-inflammatory phenotype M2, as well as reductions in pro-inflammatory cytokines and the intracellular iron concentration. Therefore, we suggest-for the first time-that nutraceuticals reduce inflammation in CCS macrophages through a novel anti-inflammatory mechanism of action, modulating iron metabolism.

The efficacy and the safety of eltrombopag in pediatric patients with severe aplastic anemia: a systematic review.

Background: Acquired aplastic anemia (AAA) in pediatric patients is a rare disorder characterized by hypocellular bone marrow and pancytopenia. Eltrombopag, an oral thrombopoietin receptor agonist, provides a hematologic improvement in adults with severe aplastic anemia (SAA) refractory to immunosuppressive therapy (IST). The association of ELT and IST was approved by the US Food and Drug Administration (FDA) for adults and children ≥2 years of age as a first-line treatment for SAA. However, the effects of ELT on pediatric patients with SAA remain controversial and limited. Methods and findings: We conducted a systematic review of the most recent literature from Pubmed, Web of Science, and Embase, published up to 20th December 2022, in order to evaluate the available evidence on the efficacy and safety of ELT added to IST for the treatment of SAA in the pediatric population. Conclusion: Eltrombopag added to the IST has shown a good safety profile, without manifestations of excessive toxic effects, although not all the results obtained from our studies support the addition of ELT to the IST in the first-line treatment of children with SAA. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/, identifier: CRD42022325859.

CNR2 functional variant (Q63R) influences childhood immune thrombocytopenic purpura.

Immune thrombocytopenic purpura is an acquired autoimmune disorder that is the most common cause of thrombocytopenia in children. The endocannabinoid system is involved in immune regulation. We evaluated a common missense variant (CAA/CGG; Q63R) of the gene encoding the cannabinoid receptor type 2 (GeneID 1269) in 190 children with immune thrombocytopenic purpura and 600 healthy controls. The allelic frequencies and genotype distribution of the polymorphism in the patients were significant compared to control samples (P=0.006 and P=0.0001, respectively). Interestingly, when acute and chronic immune thrombocytopenic purpura patients were analyzed separately with respect to controls, a significant overrepresentation of the RR genotype and of the R allele was observed only for the chronic form (P=0.00021 and P=0.011, respectively). The relative odds ratio suggested the risk of developing chronic form was more than double in immune thrombocytopenic purpura children homozygous for the variant (odds ratio=2.349, 95% CI: 1.544-3.573; P<0.001).

p27Kip1, an Intrinsically Unstructured Protein with Scaffold Properties.

The Cyclin-dependent kinase (CDK) regulator p27Kip1 is a gatekeeper of G1/S transition. It also regulates G2/M progression and cytokinesis completion, via CDK-dependent or -independent mechanisms. Recently, other important p27Kip1 functions have been described, including the regulation of cell motility and migration, the control of cell differentiation program and the activation of apoptosis/autophagy. Several factors modulate p27Kip1 activities, including its level, cellular localization and post-translational modifications. As a matter of fact, the protein is phosphorylated, ubiquitinated, SUMOylated, O-linked N-acetylglicosylated and acetylated on different residues. p27Kip1 belongs to the family of the intrinsically unstructured proteins and thus it is endowed with a large flexibility and numerous interactors, only partially identified. In this review, we look at p27Kip1 properties and ascribe part of its heterogeneous functions to the ability to act as an anchor or scaffold capable to participate in the construction of different platforms for modulating cell response to extracellular signals and allowing adaptation to environmental changes.