Pharmacological approches for the treatment of the Rett syndrome

2012/2013RIASSUNTO La syndrome di Rett (RTT) è una patologia dello sviluppo neuronale postnatale causata dalle mutazioni del gene MeCP2, situato nel cromosoma X, codificante per la Methyl CpG binding protein 2, un modulatore della trascrizione. La forma classica si manifesta in 1:10,000 bambine ed è caratterizzata da una progressiva regressione generale fisica e mentale, in seguito ad un normale sviluppo nei primi 2 anni di vita. Molti degli aspetti della patologia sono stati riprodotti in diversi modelli murini deleti per il gene MeCP2 (MeCP2-/y), inclusi la riduzione della massa cerebrale, l’atrofia neuronale e le disfunzioni cardiorespiratorie, che costituiscono i parametri più robusti e riproducibili tra i diversi modelli murini, accanto ai meno conservati parametri comportamentali, come l’ansia, la socievolezza e l’aspetto motorio. Il fenotipo Rett è caratterizzato inoltre da una riduzione dei livelli di espressione della serotonina (5HT), norepinefrina (NE) e del BDNF (Brain Derived Neurotrophic Factor). Tuttavia, è noto che i farmaci antidepressivi sono in grado di modulare i livelli di BDNF in parte regolando il sistema monoaminergico. Lo scopo di questo lavoro consiste perciò nel valutare gli effetti del trattamento cronico con antidepressivi in un modello della sindrome di Rett. Abbiamo scelto la Desipramina (DMI) come farmaco di controllo, dal momento che è già stata precedentemente utilizzata per un trial clinico della sindrome di Rett. La Desipramina è un antidepressivo che blocca il recupero di 5HT e NE a livello dello spazio sinaptico, tuttavia presenta delle complicanze cliniche a livello cardiaco. Per evitare tale effetto collaterale della DMI, abbiamo selezionato un antidepressivo altamente tollerabile, la Mirtazapina (MIR), un antagonista degli α2 autorecettori ed eterorecettori centrali e uno specifico inibitore dei recettori 5HT2 e 5HT3. Il lavoro si divide in 4 fasi: Fase 1: analisi degli effetti del trattamento con antidepressivi sul peso del corpo e del cervello ed analisi della morfologia dei neuroni piramidali della corteccia somatosensoriale in un modello murino della sindrome di Rett Fase 2: analisi degli effetti del trattamento con antidepressivi sui parametri vitali, inclusi il battito cardiaco e la frequenza respiratoria nel modello murino della sindrome di Rett Fase 3: analisi degli effetti del trattamento con antidepressivi sul comportamento nel modello murino della sindrome di Rett Fase 4: analisi degli effetti del trattamento con antidepressivi sul livello di espressione del BDNF Fase 1: analisi degli effetti del trattamento con antidepressivi sul peso del corpo e del cervello ed analisi della morfologia dei neuroni piramidali della corteccia somatosensoriale in un modello murino della sindrome di Rett Prima di tutto abbiamo valutato le caratteristiche generali del modello murino della sindrome di Rett (MeCP2-/y), osservando che il peso del corpo e del cervello dell’animale era significativamente ridotto a 42 giorni dalla nascita. Inoltre, come osservato in precedenza (Kishi and Macklis, 2004, Fukuda et al., 2005), abbiamo confermato la significativa riduzione dello spessore totale della corteccia somatosensoriale (la più compromessa in questa patologia), in particolare degli strati II-III e VI a 42 giorni dalla nascita. Abbiamo quindi trattato gli animali per due settimane a partire dal 28° giorno dalla nascita con DMI alla concentrazione 10 mg/Kg e con MIR a due differenti concentrazioni (10 o 50 mg/Kg) ed analizzato gli effetti del trattamento sul peso del corpo e del cervello. Non abbiamo riscontrato differenze per quanto riguarda il peso del corpo dopo trattamento farmacologico, tuttavia abbiamo notato un significativo aumento del peso del cervello in topi MeCP2-/y dopo 2 settimane di trattamento con MIR 50 mg/Kg, confrontato con il peso del cervello di topi MeCP2-/y della stessa età non trattati. Per meglio definire le strutture coinvolte nel recupero del peso cerebrale dopo trattamento con MIR 50 mg/Kg, abbiamo effettuato una colorazione Nissl su sezioni coronali di cervello di topo e abbiamo analizzato l’ippocampo e la corteccia somatosensoriale. Abbiamo osservato che non c’erano differenze nelle proporzioni di ogni strato ippocampale rispetto allo spessore totale dell’ippocampo lungo l’asse rostro-caudale. Tuttavia, l’analisi della corteccia somatosensoriale ha rivelato che il trattamento con DMI 10 mg/Kg e MIR 50 mg/Kg fa recuperare lo spessore totale della corteccia in topi MeCP2-/y a 42 giorni dalla nascita ed in particolare lo spessore degli strati II-III e VI che sono principalmente compromessi nel modello murino della sindrome di Rett (Kishi and Macklis, 2004, Fukuda et al., 2005). Per avere maggiori informazioni sull’effetto della MIR 50 mg/Kg a livello dei neuroni corticali, abbiamo esaminato la morfologia dei neuroni piramidali dello strato II-III della corteccia somatosensoriale in topi MeCP2-/y a 42 giorni dalla nascita utilizzando la colorazione di Golgi. Abbiamo osservato che il trattamento con MIR 50 mg/Kg induce un recupero dei deficit morfologici presenti nel modello murino (Kishi and Macklis, 2004, Fukuda et al., 2005) inclusi, la ridotta area del soma, il diametro ridotto dei dendriti apicali, l’atrofia dell’albero dendritico apicale ed in particolare quello basale, il numero delle spine “stubby” sia nei dendriti secondari apicali che basali. Infine, dal momento che è stato precedentemento osservato un deficit di rilascio del GABA in topi MeCP2-/y (Chao et al., 2010), abbiamo deciso di valutare se la MIR 50 mg/Kg era in grado di recuperare questo deficit. Abbiamo quindi dimostrato che le correnti GABA sono parzialmente recuperate dopo trattamento con MIR 50 mg/Kg nella corteccia di topi MeCP2-/y a 42 giorni dalla nascita. Fase 2: analisi degli effetti del trattamento con antidepressivi sui parametri vitali, inclusi il battito cardiaco e la frequenza respiratoria nel modello murino della sindrome di Rett I pazienti Rett e i topi MeCP2-/y presentano alterazioni cardiache e un respiro anomalo allo stato avanzato della patologia. Attraverso uno strumento non invasivo (MouseOX), abbiamo raccolto i dati relativi alla saturazione dell’ossigeno (percentuale di siti dell’emoglobina occupati dalle molecole di ossigeno), il battito cardiaco e la frequenza respiratoria (numero di battiti e respiri al minuto) e la distensione dell’arteria in base al battito cardiaco in topi Wild Type e MeCP2-/y non trattati e trattati con DMI 10 mg/Kg or MIR 50 mg/Kg. Abbiamo osservato che non ci sono alterazioni nella saturazione dell’ossigeno, tuttavia la frequenza dei battiti cardiaci e del respiro, che è ridotta nei topi MeCP2-/y non trattati, viene recuperata in seguito a trattamento con gli antidepressivi, in particolare con la MIR. Inoltre, l’effetto negativo sulla distensione dell’arteria osservato per la DMI 10 mg/Kg, non viene alterato dal trattamento con MIR 50 mg/Kg. Fase 3: analisi degli effetti del trattamento con antidepressivi sul comportamento nel modello murino della sindrome di Rett I topi MeCP2-/y sono caratterizzati dal disturbi motori e una ridotta ansia (Chahrour and Zoghbi, 2007), cosi abbiamo deciso di testare gli effetti degli antidepressivi sul comportamento del modello murino della sindrome di Rett. Attraverso il test dell’”open field”, abbiamo dimostrato che i topi MeCP2-/y trattati con i farmaci trascorrono la maggior parte del tempo del test immobili, e la loro attività in termini di capacità di alzarsi e tenersi sulle zampe posteriori e di cura personale è ridotta. Queste osservazioni sono probabilmente dovute all’effetto sedativo indotto dal trattamento con antidepressivi. Tuttavia, l’ansia che è ridotta nei topi MeCP2-/y non trattati osservata nel test dell’”elevated plus maze”, ritorna a valori normali dopo trattamento con gli antidepressivi. Fase 4: analisi degli effetti del trattamento con antidepressivi sul livello di espressione del BDNF Precedenti studi hanno dimostrato che il livello di espressione del BDNF totale è significativamente ridotto nel cervello dei topi MeCP2-/y (Chang et al., 2006, Wang et al., 2006). In questo lavoro abbiamo dapprima dimostrato come i livelli delle diverse isoforme del BDNF variano sulla base della mutazione del gene MeCP2 nei pazienti Rett. Successivamente abbiamo valutato le diverse isoforme del BDNF nel prosencefalo di topi MeCP2-/y dimostrando come esse siano significativamente ridotte a 42 giorni dalla nascita. Tuttavia, il trattamento con DMI 10 mg/Kg e MIR 50 mg/Kg non è in grado di recuperare in modo significativo il livello di mRNA. Abbiamo quindi valutato il livello proteico del BDNF, dimostrando un aumento della neurotrofina a livello corticale e una diminuzione a livello ippocampale in topi MeCP2-/y non trattati ma non statisticamente significativo. Tuttavia il trattamento con MIR 50 mg/Kg sembra recuperare il livello del BDNF, sebbene non sia significativo.  ABSTRACT Rett syndrome (RTT) is an X-linked postnatal neurodevelopmental disorder caused by the mutations on MeCP2 gene which encodes for the Methyl CpG binding protein 2, a transcriptional regulator. The classical form manifests in girls with an incidence of 1:10,000 with a progressive general physical and mental regression after a normal development during the first two years of age. Several clinical features are recapitulated in MeCP2-/y mice, including the reduced brain mass, neuronal atrophy and the cardiorespiratory abnormalities, which are considered the most robust and reproducible parameters among the Rett mouse models and the less conserved alterations on mice behavior. Rett phenotype was characterized by a reduction on serotonin, norepinephrine (5HT; NE) and BDNF (Brain Derived Neurotrophic Factor) expression level. However, it is known that the antidepressants drugs modulate BDNF expression level partly by regulation of monoamine systems. The aim of the project is to evaluate the effects of repeated antidepressant treatments in a Rett mouse model. We choose Desipramine (DMI) as control drug because it was previously used in a clinical trial of Rett syndrome. DMI blocks the reuptake of 5HT and NE, but it has some cardiac complications. To overcame the cardiac side effect of DMI, we selected the highly tolerable antidepressant Mirtazapine (MIR), which is an antagonist of central α2 autoreceptors and α2 heteroreceptors and a specific blocker of 5HT2 and 5HT3 receptors. The project comprises four phases: Phase1: Analysis of the effects of antidepressant treatments on body and brain weight, including the morphology of the somatosensory pyramidal neurons in a model of Rett syndrome (MeCP2-/y) Phase2: Analysis of the effects of antidepressant treatments on the vital signs parameters, including heart and breath rate in MeCP2-/y mice Phase3: Analysis of the effects of antidepressant treatments on the behavior of the mice (open field and plus maze test) in MeCP2-/y mice Phase4: Analysis of the effects of antidepressant treatments on brain derived neurotrophic factor (BDNF) expression level Phase1: Analysis of the effects of the drugs on body and brain weight, including the morphology of the somatosensory pyramidal neurons in a model of Rett syndrome (MeCP2-/y) First of all, we evaluated the general features of the Rett mouse model, observing that the body and the brain weight of MeCP2-/y mice were reduced at postnatal day 42 (p42). We found also that there is a significant reduction on total cortical thickness, in particular of layers II-III and VI at p42 as observed in previous studies (Kishi and Macklis, 2004, Fukuda et al., 2005). Then, we analyzed the effects of DMI 10 mg/Kg and MIR (at two different concentration: 10 or 50 mg/Kg) treatments on body and brain weight. No difference was observed for body weight, while an increase in brain weight was noticed after treatment with MIR 50 mg/Kg in p42 MeCP2-/y mice compared to MeCP2-/y untreated mice. To better define the brain structures involved in the rescue of the brain weight after MIR 50 mg/Kg treatment, we performed a Nissl staining and we analyzed the hippocampus and the somatosensory cortex. We found that among p42 MeCP2-/y treated mice, there were no differences in the proportion of each hippocampal layer to the total thickness along the rostro-caudal axis. However, the analysis of the somatosensory cortex revealed that DMI 10 mg/Kg and MIR 50 mg/Kg rescued the total cortical thickness in p42 MeCP2-/y mice and in particular the layers II-III and VI which are principally compromised in Rett mouse model (Kishi and Macklis, 2004, Fukuda et al., 2005). To gain further insight regarding the effect of Mirtazapine treatment on cortical neurons, we investigated the morphology of layer II-III pyramidal neurons of the somatosensory cortex in MeCP2-/y mice using Golgi staining. We observed that MIR 50 mg/Kg treatment was able to recover the neuronal morphology deficits of p42 MeCP2-/y mice (Kishi and Macklis, 2004, Fukuda et al., 2005), including, the small soma area, the reduced diameter of apical dendrites, the atrophy of apical and, in particular, the basal dendritic arborization, the number of secondary basal dendrites, the number of stubby spines both in secondary apical and basal dendrites. Finally, as a deficit on GABA release in MeCP2-/y mice was previously described (Chao et al., 2010), we investigatd if Mirtazapine could rescue this deficit. Indeed, we found that GABA currents were rescued by MIR 50 mg/Kg treatment in the cortex of p42 MeCP2-/y mice, although without reaching full recovery. Phase2: Analysis of the effects of the drugs on the vital signs parameters, including heart and breath rate in MeCP2-/y mice Rett patients and MeCP2-/y mice presents cardiac alterations and breathing abnormalities in a later stage of the disorder. Through a non-invasive instrument (MouseOX) we collected the data regarding the Oxygen Saturation (percentage of sites of arterial hemoglobin occupied by oxygen molecules), the Hearth and the Breath Rate (number of beats or breaths per minute) and the Pulse Distention (change in distension of the arterial blood vessels due to a cardiac pulse) on Wild Type and MeCP2-/y mice untreated or treated with DMI 10 mg/Kg or MIR 50 mg/Kg. We found that no alterations was observed for the oxygen saturation, however the frequency of heart and breath are rescued after drug treatments. A negative effect of Desipramine was observed in pulse distention which is not affected with Mirtazapine treatment. Phase3: Analysis of the effects of the drugs on the behavior of the mice (open field and plus maze test) MeCP2-/y mice are characterized by motor abnormalities and a decreased anxiety (Chahrour and Zoghbi, 2007), thus, we tested the effects of the antidepressant drugs on the behavior of MeCP2-/y mice. Through an open field test, we found that the MeCP2-/y mice treated with the drugs spent more of the time immobile, and their activity in terms of number of rearing and grooming was reduced. These observations are probably due to the sedative effect of antidepressant treatments. However, the anxiety was recover to normal levels in MeCP2-/y mice treated with the antidepressants in the elevated plus maze. Phase4: Analysis of the effects of treatments on BDNF expression level Previous studies showed that total BDNF expression level was significantly reduced in the brain of MeCP2-/y mice (Chang et al., 2006, Wang et al., 2006). First of all, we demonstrated that the levels of BDNF isoforms depend on mutations in MeCP2 gene in Rett patients. Then, we evaluated the BDNF splice variants in the forebrain of MeCP2-/y mice and we demonstrated that they were significantly reduced at p42. However, treatments with DMI 10 mg/Kg or MIR 50 mg/Kg not rescue significantly the mRNA of BDNF. Therefore, we evaluated the protein level of BDNF and we demonstrated a no statistically significant increase of the neurotrophin in the cortex and a decrease in the hippocampus in MeCP2-/y untreated mice. However, the treatment with MIR 50 mg/Kg seemed to rescue the protein level of BDNF, even if no statistically significant.XXVI Ciclo198

Pharmacological treatment with mirtazapine rescues cortical atrophy and respiratory deficits in MeCP2 null mice

Loss of MeCP2 (Methyl CpG binding protein 2) in Rett syndrome (RTT) causes brain weight decrease, shrinkage of the cortex with reduced dendritic arborization, behavioral abnormalities, seizures and cardio-respiratory complications. The observed monoamine neurotransmitters reduction in RTT suggested antidepressants as a possible therapy. We treated MeCP2-null mice from postnatal-day 28 for two weeks with desipramine, already tested in RTT, or mirtazapine, an antidepressant with limited side-effects, known to promote GABA release. Mirtazapine was more effective than desipramine in restoring somatosensory cortex thickness by fully rescuing pyramidal neurons dendritic arborization and spine density. Functionally, mirtazapine treatment normalized heart rate, breath rate, anxiety levels, and eliminated the hopping behavior observed in MeCP2-null mice, leading to improved phenotypic score. These morphological and functional effects of mirtazapine were accompanied by reestablishment of the GABAergic and glutamatergic receptor activity recorded in cortex and brainstem tissues. Thus, mirtazapine can represent a new potential pharmacological treatment for the Rett syndrome

Targeted chitosan nanobubbles as a strategy to down-regulate microRNA-17 into B-cell lymphoma models

IntroductionMicroRNAs represent interesting targets for new therapies because their altered expression influences tumor development and progression. miR-17 is a prototype of onco-miRNA, known to be overexpressed in B-cell non-Hodgkin lymphoma (B-NHL) with peculiar clinic-biological features. AntagomiR molecules have been largely studied to repress the regulatory functions of up-regulated onco-miRNAs, but their clinical use is mainly limited by their rapid degradation, kidney elimination and poor cellular uptake when injected as naked oligonucleotides.MethodsTo overcome these problems, we exploited CD20 targeted chitosan nanobubbles (NBs) for a preferential and safe delivery of antagomiR17 to B-NHL cells.ResultsPositively charged 400 nm-sized nanobubbles (NBs) represent a stable and effective nanoplatform for antagomiR encapsulation and specific release into B-NHL cells. NBs rapidly accumulated in tumor microenvironment, but only those conjugated with a targeting system (antiCD20 antibodies) were internalized into B-NHL cells, releasing antagomiR17 in the cytoplasm, both in vitro and in vivo. The result is the down-regulation of miR-17 level and the reduction in tumor burden in a human-mouse B-NHL model, without any documented side effects.DiscussionAnti-CD20 targeted NBs investigated in this study showed physico-chemical and stability properties suitable for antagomiR17 delivery in vivo and represent a useful nanoplatform to address B-cell malignancies or other cancers through the modification of their surface with specific targeting antibodies

IGLV3-21*01 is an inherited risk factor for CLL through the acquisition of a single-point mutation enabling autonomous BCR signaling

The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors

Age-dependent pattern of cerebellar susceptibility to bilirubin neurotoxicity in vivo in mice

Neonatal jaundice is caused by high levels of unconjugated bilirubin. It is usually a temporary condition caused by delayed induction of UGT1A1, which conjugates bilirubin in the liver. To reduce bilirubin levels, affected babies are exposed to phototherapy (PT), which converts toxic bilirubin into water-soluble photoisomers that are readily excreted out. However, in some cases uncontrolled hyperbilirubinemia leads to neurotoxicity. To study the mechanisms of bilirubin-induced neurological damage (BIND) in vivo, we generated a mouse model lacking the Ugt1a1 protein and, consequently, mutant mice developed jaundice as early as 36 hours after birth. The mutation was transferred into two genetic backgrounds (C57BL/6 and FVB/NJ). We exposed mutant mice to PT for different periods and analyzed the resulting phenotypes from the molecular, histological and behavioral points of view. Severity of BIND was associated with genetic background, with 50% survival of C57BL/6‑Ugt1−/− mutant mice at postnatal day 5 (P5), and of FVB/NJ-Ugt1−/− mice at P11. Life-long exposure to PT prevented cerebellar architecture alterations and rescued neuronal damage in FVB/NJ-Ugt1−/− but not in C57BL/6-Ugt1−/− mice. Survival of FVB/NJ-Ugt1−/− mice was directly related to the extent of PT treatment. PT treatment of FVB/NJ-Ugt1−/− mice from P0 to P8 did not prevent bilirubin-induced reduction in dendritic arborization and spine density of Purkinje cells. Moreover, PT treatment from P8 to P20 did not rescue BIND accumulated up to P8. However, PT treatment administered in the time-window P0–P15 was sufficient to obtain full rescue of cerebellar damage and motor impairment in FVB/NJ-Ugt1−/− mice. The possibility to modulate the severity of the phenotype by PT makes FVB/NJ-Ugt1−/− mice an excellent and versatile model to study bilirubin neurotoxicity, the role of modifier genes, alternative therapies and cerebellar development during high bilirubin conditions

KRAS and RAS-MAPK Pathway Deregulation in Mature B Cell Lymphoproliferative Disorders

KRAS mutations account for the most frequent mutations in human cancers, and are generally correlated with disease aggressiveness, poor prognosis, and poor response to therapies. KRAS is required for adult hematopoiesis and plays a key role in B cell development and mature B cell proliferation and survival, proved to be critical for B cell receptor-induced ERK pathway activation. In mature B cell neoplasms, commonly seen in adults, KRAS and RAS-MAPK pathway aberrations occur in a relevant fraction of patients, reaching high recurrence in some specific subtypes like multiple myeloma and hairy cell leukemia. As inhibitors targeting the RAS-MAPK pathway are being developed and improved, it is of outmost importance to precisely identify all subgroups of patients that could potentially benefit from their use. Herein, we review the role of KRAS and RAS-MAPK signaling in malignant hematopoiesis, focusing on mature B cell lymphoproliferative disorders. We discuss KRAS and RAS-MAPK pathway aberrations describing type, incidence, mutual exclusion with other genetic abnormalities, and association with prognosis. We review the current therapeutic strategies applied in mature B cell neoplasms to counteract RAS-MAPK signaling in pre-clinical and clinical studies, including most promising combination therapies. We finally present an overview of genetically engineered mouse models bearing KRAS and RAS-MAPK pathway aberrations in the hematopoietic compartment, which are valuable tools in the understanding of cancer biology and etiology

Multiple Mechanisms of NOTCH1 Activation in Chronic Lymphocytic Leukemia: NOTCH1 Mutations and Beyond

The Notch signaling pathway plays a fundamental role for the terminal differentiation of multiple cell types, including B and T lymphocytes. The Notch receptors are transmembrane proteins that, upon ligand engagement, undergo multiple processing steps that ultimately release their intracytoplasmic portion. The activated protein ultimately operates as a nuclear transcriptional co-factor, whose stability is finely regulated. The Notch pathway has gained growing attention in chronic lymphocytic leukemia (CLL) because of the high rate of somatic mutations of the NOTCH1 gene. In CLL, NOTCH1 mutations represent a validated prognostic marker and a potential predictive marker for anti-CD20-based therapies, as pathological alterations of the Notch pathway can provide significant growth and survival advantage to neoplastic clone. However, beside NOTCH1 mutation, other events have been demonstrated to perturb the Notch pathway, namely somatic mutations of upstream, or even apparently unrelated, proteins such as FBXW7, MED12, SPEN, SF3B1, as well as physiological signals from other pathways such as the B-cell receptor. Here we review these mechanisms of activation of the NOTCH1 pathway in the context of CLL; the resulting picture highlights how multiple different mechanisms, that might occur under specific genomic, phenotypic and microenvironmental contexts, ultimately result in the same search for proliferative and survival advantages (through activation of MYC), as well as immune escape and therapy evasion (from anti-CD20 biological therapies). Understanding the preferential strategies through which CLL cells hijack NOTCH1 signaling may present important clues for designing targeted treatment strategies for the management of CLL

TP53 mutations with low variant allele frequency predict short survival in chronic lymphocytic leukemia

Purpose: In chronic lymphocytic leukemia (CLL), TP53 mutations are associated with reduced survival and resistance to standard chemoimmunotherapy (CIT). Nevertheless, the clinical impact of subclonal TP53 mutations below 10% to 15% variant allele frequency (VAF) remains unclear. Experimental Design: Using a training/validation approach, we retrospectively analyzed the clinical and biological features of TP53 mutations above (high-VAF) or below (low-VAF) the previously reported 10.0% VAF threshold, as determined by deep next-generation sequencing. Clinical impact of low-VAF TP53 mutations was also confirmed in a cohort (n = 251) of CLL treated with fludarabine-cyclophosphamide-rituximab (FCR) or FCR-like regimens from two UK trials. Results: In the training cohort, 97 of 684 patients bore 152 TP53 mutations, while in the validation cohort, 71 of 536 patients had 109 TP53 mutations. In both cohorts, patients with the TP53 mutation experienced significantly shorter overall survival (OS) than TP53 wild-type patients, regardless of the TP53 mutation VAF. By combining TP53 mutation and 17p13.1 deletion (del17p) data in the total cohort (n = 1,220), 113 cases were TP53 mutated only (73/113 with low-VAF mutations), 55 del17p/TP53 mutated (3/55 with low-VAF mutations), 20 del17p only, and 1,032 (84.6%) TP53 wild-type. A model including low-VAF cases outperformed the canonical model, which considered only high-VAF cases (c-indices 0.643 vs. 0.603, P < 0.0001), and improved the prognostic risk stratification of CLL International Prognostic Index. Clinical results were confirmed in CIT-treated cases (n = 552) from the retrospective cohort, and the UK trials cohort. Conclusions: TP53 mutations affected OS regardless of VAF. This finding can be used to update the definition of TP53 mutated CLL for clinical purposes