NOTCH1 inhibition regulates evolutionary conserved miRNAs in T-cell Acute Lymphoblastic Leukemia (T-ALL)

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic tumor, resulting from the transformation of T-cell progenitors. Activating mutations in the NOTCH1 ligand-activated transcription factor oncogene are found in over 60% of T-ALL cases. Recently, several microRNAs have been shown to cooperate with NOTCH1 in the pathogenesis of T-ALL. However, little is currently known on the microRNAs that are regulated following NOTCH1 inhibition. Thus, in view of future therapies that may combine NOTCH1 inhibition with microRNA based therapy we pursued to study the microRNAs regulated following NOTCH1 inhibition and their functional role in T-ALL pathogenesis. We first generated a mouse model of NOTCH1-induced leukemia, that carries a NOTCH1 mutation recurrently found in human T-ALL patients (L1601P-PEST), and inhibited NOTCH1 signaling in vivo by treating diseased animals with a potent gamma secretase inhibitor (DBZ). These NOTCH1-induced T-ALL samples were subjected to miRNA profiling using a mouse array (8X60K release 19.0; Agilent) and, in parallel, gene expression analysis using SurePrint G3 Mouse Gene Expression v2 array (Agilent). The MYC and NOTCH signatures resulted strongly down-regulated following NOTCH1 inhibition by Gene Set Enriched Analysis (GSEA) demonstrating the efficacy of our experimental model. Among the NOTCH1 down-regulated miRNAs, we found the miR-17-92 cluster, previously reported to be highly expressed in T-ALL samples. Their regulation was also confirmed in another mouse model of NOTCH1-induced T-ALL and in human T-leukemia cells. Notably, we identified miR-34a-5p, miR-22a-3p and miR-199a-5p to be significantly up-regulated following NOTCH1-inhibition suggesting a putative role as tumor suppressors in NOTCH1-driven leukemia. Even if we can hypothesize that these miRNAS could play an important role in murine T-cell leukemia, they resulted not expressed in human T-ALL cells. Differently, miR-22a-3p resulted significantly up-regulated following NOTCH1 inhibition both in mouse and human T-ALL cells. Moreover, the overexpression of miR-22a-3p inhibited in vitro colony formation in T-ALL cell lines carrying constitutive NOTCH1 activation and significantly impaired tumor growth in vivo when overexpressed in human T-ALL cells, suggesting a tumor suppressor role for miR-22 in T-ALL downstream of NOTCH1. Meta-analysis from the human T-ALL dataset already published showed, amongst the significantly up-regulated gene sets, targets of the microRNAs belonging to the miR-17/92 cluster. These results are in accordance with our murine microRNA differential expression analysis, in which we found components of miR-17/92 cluster to be strongly down-regulated. Moreover, using the same human dataset, we ran GSEA against the C3 sub collection of mir targets in the MSigDB v6.0, including additional gene sets with putative targets of miR-22. Amongst the downregulated gene sets, we identified 23 down-regulated genes that were consistently contributing to the negative enrichment of all the three selected gene sets of miR-22 targets. Amongst these genes, we found the Peroxisome Proliferator-Activated Receptor Gamma, Coactivator 1 Beta (PGC-1β), that is involved in mitochondrial metabolism. Notably, GSEA analysis, identified transcription factors significantly regulated upon treatment with DBZ in human T-ALL cells, finding that the PPARG_01 gene set, containing targets of the PPARG transcription factor, was significantly down-regulated in this context. PGC-1β resulted significantly down-regulated following NOTCH1 inhibition in vivo in human PDTALL xenografts and in one of three miR-22 overexpressing T-ALL cell lines at transcription level. On the other hand, the amount of PGC-1b protein was found to be very high and insensitive to NOTCH1 inhibition or miR-22 overexpression. In conclusion, we found that miR-22-3p was down-regulated in T-ALL cells and its expression level could be restored following NOTCH1 inhibition. miR-22-3p over-expression affected in vivo tumor growth, possibly altering homing to supportive niches and so favouring disease progression, supporting its tumor suppressor role in NOTCH1-mutated T-ALL cells. Meta-analysis of NOTCH1 regulated genes in human T-ALL cell lines indicated PGC-1β as a putative miR-22 target gene, whose expression appeared significantly regulated only at the transcriptional level, while protein level was found unaltered. Thus, we are still investigating on the role of NOTCH1/has-miR-22/PPARG axis because understanding the mechanism of action of miR-22-3p in T-ALL cells could be contribute to the successful treatment for T-ALL, opening possibilities of future therapeutic interventions that may combine NOTCH1 inhibition with microRNA based therapy.La leucemia acuta linfoblastica di tipo T (LAL-T) è un tumore ematologico aggressivo, risultante dalla trasformazione dei progenitori delle cellule T. Le mutazioni attivanti nel fattore di trascrizione oncogene NOTCH1 attivato dal ligando si trovano in oltre il 60% dei casi della LAL-T. Recentemente, è stato dimostrato che diversi microRNAs cooperano con NOTCH1 nella patogenesi della LAL-T. Tuttavia, attualmente poco è noto sui microRNAs regolati in seguito all'inibizione di NOTCH1. Quindi, in visione di future terapie che potrebbero combinare l'inibizione di NOTCH1 con la terapia basata sui microRNAs, abbiamo intrapreso lo studio sui microRNAs regolati in seguito all'inibizione di NOTCH1 e sul loro ruolo funzionale nella patogenesi della LAL-T. Dapprima, abbiamo generato un modello murino di leucemia indotta da NOTCH1, che porta una mutazione in NOTCH1 frequentemente trovata in pazienti con LAL-T (L1601P-PEST), ed abbiamo inibito la segnalazione di NOTCH1 in vivo trattando animali malati con un potente inibitore della gamma secretasi (DBZ ). Questi campioni di LAL-T indotta da NOTCH1 sono stati sottoposti all’analisi di espressione dei miRNAs utilizzando un mouse array (8X60K release 19.0; Agilent) e, in parallelo, all’analisi di espressione genica utilizzando SurePrint G3 Mouse Gene Expression v2 array (Agilent). I geni a valle di MYC e di NOTCH sono risultati significativamente repressi a seguito dell'inibizione di NOTCH1 grazie alla Gene Set Enriched Analysis (GSEA), che abbiamo trovato il cluster miR-17-92, precedentemente riportato come altamente espresso nei campioni di LAL-T. La loro regolazione è stata confermata anche in un altro modello murino di LAL-T indotta da NOTCH1 e nelle cellule umane di leucemia di tipo T. In particolare, abbiamo identificato i miR-34a-5p, miR-22a-3p e miR-199a-5p per essere significativamente indotti inseguito all'inibizione di NOTCH1 suggerendo un loro presunto ruolo come soppressori del tumore nella leucemia indotta da NOTCH1. Anche se possiamo ipotizzare che questi miRNAs possano svolgere un ruolo importante nella leucemia di tipo T murina, non sono risultati essere espressi nelle cellule di LAL-T umane. Diversamente, il miR-22a-3p risultava significativamente indotto in seguito all'inibizione di NOTCH1 sia nelle cellule di LAL-T murine che umane. Inoltre, la sovra-espressione del miR-22a-3p ha inibito la formazione di colonie in vitro in linee cellulari di LAL-T, che portavano l’attivazione costitutiva di NOTCH1, e ha significativamente ridotto la crescita del tumore in vivo quando sovra-espresso in cellule umane di LAL-T, suggerendo un ruolo come soppressore del tumore per il miR-22 nella LAL-T a valle di NOTCH1. La meta-analisi del dataset umano di LAL-T già pubblicato ha mostrato, tra i gruppi di geni significativamente indotti, i target dei microRNAs appartenenti al cluster miR-17/92. Questi risultati sono in accordo con la nostra analisi di espressione differenziale dei microRNAs murini, in cui abbiamo trovato che i componenti del cluster miR-17/92 sono fortemente repressi. Inoltre, utilizzando lo stesso set di dati umani, abbiamo eseguito una GSEA verso la C3 sottoclasse dei miR targets in MSigDB v6.0, inclusi set di geni aggiuntivi con putativi targets del miR-22. Tra i gruppi di geni repressi, abbiamo identificato 23 geni repressi che contribuivano in modo consistente all'arricchimento negativo di tutti e tre i gruppi di geni selezionati dei bersagli del miR-22. Tra questi geni, abbiamo trovato il Recettore Gamma attivato dal Proliferatore del Perossisoma, Coattivatore 1 Beta (PGC-1β), coinvolto nel metabolismo mitocondriale. In particolare, l'analisi GSEA ha identificato i fattori di trascrizione regolati in modo significativo dopo il trattamento con DBZ in cellule umane di LAL-T, trovando che il set di geni PPARG_01, contenente bersagli del fattore di trascrizione PPARG, era significativamente represso in questo contesto. PGC-1β è risultato significativamente represso in seguito all'inibizione di NOTCH1 in vivo negli xenografts di PDTALL umani e in una delle tre linee cellulari di LAL-T, che sovraesprimono il miR-22, a livello trascrizionale. D'altra parte, la quantità di proteina PGC-1β è risultata essere molto elevata e insensibile all'inibizione di NOTCH1 o alla sovraespressione del miR-22. In conclusione, abbiamo scoperto che il miR-22-3p era represso nelle cellule di LAL-T e il suo livello di espressione poteva essere ripristinato inseguito all'inibizione di NOTCH1. La sovraespressione del miR-22-3p influenzava la crescita tumorale in vivo, probabilmente alterando la migrazione e favorendo così la progressione della malattia, supportando il suo ruolo di soppressore del tumore nelle cellule di LAL-T con NOTCH1 mutato. La meta-analisi dei geni regolati da NOTCH1 nelle linee cellulari umane di LAL-T indicava PGC-1β come un possibile gene bersaglio del miR-22, la cui espressione appariva significativamente regolata solo a livello trascrizionale, mentre a livello proteico risultava inalterata. Pertanto, stiamo ancora studiando il ruolo dell'asse NOTCH1 / hsa-miR-22 / PPARG perché la comprensione del meccanismo d'azione di miR-22-3p nelle cellule di LAL-T potrebbe contribuire al successo del trattamento per la LAL-T, aprendo possibilità di futuri interventi terapeutici che possono combinare l'inibizione di NOTCH1 con la terapia basata su microRNAs

NOTCH1 inhibition regulates evolutionary conserved miRNAs in T-cell Acute Lymphoblastic Leukemia (T-ALL)

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic tumor, resulting from the transformation of T-cell progenitors. Activating mutations in the NOTCH1 ligand-activated transcription factor oncogene are found in over 60% of T-ALL cases. Recently, several microRNAs have been shown to cooperate with NOTCH1 in the pathogenesis of T-ALL. However, little is currently known on the microRNAs that are regulated following NOTCH1 inhibition. Thus, in view of future therapies that may combine NOTCH1 inhibition with microRNA based therapy we pursued to study the microRNAs regulated following NOTCH1 inhibition and their functional role in T-ALL pathogenesis. We first generated a mouse model of NOTCH1-induced leukemia, that carries a NOTCH1 mutation recurrently found in human T-ALL patients (L1601P-PEST), and inhibited NOTCH1 signaling in vivo by treating diseased animals with a potent gamma secretase inhibitor (DBZ). These NOTCH1-induced T-ALL samples were subjected to miRNA profiling using a mouse array (8X60K release 19.0; Agilent) and, in parallel, gene expression analysis using SurePrint G3 Mouse Gene Expression v2 array (Agilent). The MYC and NOTCH signatures resulted strongly down-regulated following NOTCH1 inhibition by Gene Set Enriched Analysis (GSEA) demonstrating the efficacy of our experimental model. Among the NOTCH1 down-regulated miRNAs, we found the miR-17-92 cluster, previously reported to be highly expressed in T-ALL samples. Their regulation was also confirmed in another mouse model of NOTCH1-induced T-ALL and in human T-leukemia cells. Notably, we identified miR-34a-5p, miR-22a-3p and miR-199a-5p to be significantly up-regulated following NOTCH1-inhibition suggesting a putative role as tumor suppressors in NOTCH1-driven leukemia. Even if we can hypothesize that these miRNAS could play an important role in murine T-cell leukemia, they resulted not expressed in human T-ALL cells. Differently, miR-22a-3p resulted significantly up-regulated following NOTCH1 inhibition both in mouse and human T-ALL cells. Moreover, the overexpression of miR-22a-3p inhibited in vitro colony formation in T-ALL cell lines carrying constitutive NOTCH1 activation and significantly impaired tumor growth in vivo when overexpressed in human T-ALL cells, suggesting a tumor suppressor role for miR-22 in T-ALL downstream of NOTCH1. Meta-analysis from the human T-ALL dataset already published showed, amongst the significantly up-regulated gene sets, targets of the microRNAs belonging to the miR-17/92 cluster. These results are in accordance with our murine microRNA differential expression analysis, in which we found components of miR-17/92 cluster to be strongly down-regulated. Moreover, using the same human dataset, we ran GSEA against the C3 sub collection of mir targets in the MSigDB v6.0, including additional gene sets with putative targets of miR-22. Amongst the downregulated gene sets, we identified 23 down-regulated genes that were consistently contributing to the negative enrichment of all the three selected gene sets of miR-22 targets. Amongst these genes, we found the Peroxisome Proliferator-Activated Receptor Gamma, Coactivator 1 Beta (PGC-1β), that is involved in mitochondrial metabolism. Notably, GSEA analysis, identified transcription factors significantly regulated upon treatment with DBZ in human T-ALL cells, finding that the PPARG_01 gene set, containing targets of the PPARG transcription factor, was significantly down-regulated in this context. PGC-1β resulted significantly down-regulated following NOTCH1 inhibition in vivo in human PDTALL xenografts and in one of three miR-22 overexpressing T-ALL cell lines at transcription level. On the other hand, the amount of PGC-1b protein was found to be very high and insensitive to NOTCH1 inhibition or miR-22 overexpression. In conclusion, we found that miR-22-3p was down-regulated in T-ALL cells and its expression level could be restored following NOTCH1 inhibition. miR-22-3p over-expression affected in vivo tumor growth, possibly altering homing to supportive niches and so favouring disease progression, supporting its tumor suppressor role in NOTCH1-mutated T-ALL cells. Meta-analysis of NOTCH1 regulated genes in human T-ALL cell lines indicated PGC-1β as a putative miR-22 target gene, whose expression appeared significantly regulated only at the transcriptional level, while protein level was found unaltered. Thus, we are still investigating on the role of NOTCH1/has-miR-22/PPARG axis because understanding the mechanism of action of miR-22-3p in T-ALL cells could be contribute to the successful treatment for T-ALL, opening possibilities of future therapeutic interventions that may combine NOTCH1 inhibition with microRNA based therapy

miR-22-3p Negatively Affects Tumor Progression in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a rare, aggressive disease arising from T-cell precursors. NOTCH1 plays an important role both in T-cell development and leukemia progression, and more than 60% of human T-ALLs harbor mutations in components of the NOTCH1 signaling pathway, leading to deregulated cell growth and contributing to cell transformation. Besides multiple NOTCH1 target genes, microRNAs have also been shown to regulate T-ALL initiation and progression. Using an established mouse model of T-ALL induced by NOTCH1 activation, we identified several microRNAs downstream of NOTCH1 activation. In particular, we found that NOTCH1 inhibition can induce miR-22-3p in NOTCH1-dependent tumors and that this regulation is also conserved in human samples. Importantly, miR-22-3p overexpression in T-ALL cells can inhibit colony formation in vitro and leukemia progression in vivo. In addition, miR-22-3p was found to be downregulated in T-ALL specimens, both T-ALL cell lines and primary samples, relative to immature T-cells. Our results suggest that miR-22-3p is a functionally relevant microRNA in T-ALL whose modulation can be exploited for therapeutic purposes to inhibit T-ALL progression

Aberrant Signaling Pathways in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease caused by the malignant transformation of immature progenitors primed towards T-cell development. Clinically, T-ALL patients present with diffuse infiltration of the bone marrow by immature T-cell blasts high blood cell counts, mediastinal involvement, and diffusion to the central nervous system. In the past decade, the genomic landscape of T-ALL has been the target of intense research. The identification of specific genomic alterations has contributed to identify strong oncogenic drivers and signaling pathways regulating leukemia growth. Notwithstanding, T-ALL patients are still treated with high-dose multiagent chemotherapy, potentially exposing these patients to considerable acute and long-term side effects. This review summarizes recent advances in our understanding of the signaling pathways relevant for the pathogenesis of T-ALL and the opportunities offered for targeted therapy

Calcineurin complex isolated from T-cell acute lymphoblastic leukemia (T-ALL) cells identifies new signaling pathways including mTOR/AKT/S6K whose inhibition synergize with Calcineurin inhibition to promote T-ALL cell death

Calcineurin (Cn) is a calcium activated protein phosphatase involved in many aspects of normal T cell physiology, however the role of Cn and/or its downstream targets in leukemogenesis are still ill-defined. In order to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-cell acute lymphoblastic leukemia (T-ALL) we used tandem affinity chromatography, followed by mass spectrometry to purify novel Cn-interacting partners. We found the Cn-interacting proteins to be part of numerous cellular signaling pathways including eIF2 signaling and mTOR signaling. Coherently, modulation of Cn activity in T-ALL cells determined alterations in the phosphorylation status of key molecules implicated in protein translation such as eIF-2\u3b1 and ribosomal protein S6. Joint targeting of PI3K-mTOR, eIF-2\u3b1 and 14-3-3 signaling pathways with Cn unveiled novel synergistic pro-apoptotic drug combinations. Further analysis disclosed that the synergistic interaction between PI3K-mTOR and Cn inhibitors was prevalently due to AKT inhibition. Finally, we showed that the synergistic pro-apoptotic response determined by jointly targeting AKT and Cn pathways was linked to down-modulation of key anti-apoptotic proteins including Mcl-1, Claspin and XIAP. In conclusion, we identify AKT inhibition as a novel promising drug combination to potentiate the pro-apoptotic effects of Cn inhibitors

Aberrant Signaling Pathways in T-Cell Acute Lymphoblastic Leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease caused by the malignant transformation of immature progenitors primed towards T-cell development. Clinically, T-ALL patients present with diffuse infiltration of the bone marrow by immature T-cell blasts high blood cell counts, mediastinal involvement, and diffusion to the central nervous system. In the past decade, the genomic landscape of T-ALL has been the target of intense research. The identification of specific genomic alterations has contributed to identify strong oncogenic drivers and signaling pathways regulating leukemia growth. Notwithstanding, T-ALL patients are still treated with high-dose multiagent chemotherapy, potentially exposing these patients to considerable acute and long-term side effects. This review summarizes recent advances in our understanding of the signaling pathways relevant for the pathogenesis of T-ALL and the opportunities offered for targeted therap

Hematemesis in Infants: The First Evidence-Based Score to Predict the Need for Timely Endoscopy

Objectives Infantile acute upper gastrointestinal bleeding involves a decision for therapeutic intervention that most pediatricians first coming into contact with the patient are, not unreasonably, unable to objectively provide. Therefore, some objective tools of individual risk assessment would seem to be crucial. The principal aim of the present study was to investigate the anamnestic and clinical parameters of infants with hematemesis, together with laboratory and instrumental findings, to create a scoring system that may help identify those infants requiring an appropriate and timely application of upper gastrointestinal (GI) endoscopy. Methods Clinical data of infants admitted for hematemesis to the participating centers over the study period were systematically collected. According to the outcome dealing with rebleeding, need for blood transfusion, mortality, finding of GI bleeding lesions, or need for surgical intervention, patients were blindly divided into a group with major clinical severity and a group with minor clinical severity. Univariate and multivariate logistic regressions were conducted to investigate significant prognostic factors for clinical severity. Results According to our findings, we drafted a practical diagnostic algorithm and a clinical score able to predict the need for timely upper GI endoscopy (BLOVO infant score). Our clinical scoring system was created by incorporating anamnestic factors, clinical parameters, and laboratory findings that emerged as predictors of a worst outcome. Conclusions We provided the first objective tool of individual risk assessment for infants with hematemesis, which could be very useful for pediatricians first coming into contact with the patient in the emergency department

Cross-talk between GLI transcription factors and FOXC1 promotes T-cell acute lymphoblastic leukemia dissemination

T-cell acute lymphoblastic leukemia (T-ALL) is a highly malignant pediatric leukemia, where few therapeutic options are available for patients which relapse. We find that therapeutic targeting of GLI transcription factors by GANT-61 is particularly effective against NOTCH1 unmutated T-ALL cells. Investigation of the functional role of GLI1 disclosed that it contributes to T-ALL cell proliferation, survival, and dissemination through the modulation of AKT and CXCR4 signaling pathways. Decreased CXCR4 signaling following GLI1 inactivation was found to be prevalently due to post-transcriptional mechanisms including altered serine 339 CXCR4 phosphorylation and cortactin levels. We also identify a novel cross-talk between GLI transcription factors and FOXC1. Indeed, GLI factors can activate the expression of FOXC1 which is able to stabilize GLI1/2 protein levels through attenuation of their ubiquitination. Further, we find that prolonged GLI1 deficiency has a double-edged role in T-ALL progression favoring disease dissemination through the activation of a putative AKT/FOXC1/GLI2 axis. These findings have clinical significance as T-ALL patients with extensive central nervous system dissemination show low GLI1 transcript levels. Further, T-ALL patients having a GLI2-based Hedgehog activation signature are associated with poor survival. Together, these findings support a rationale for targeting the FOXC1/AKT axis to prevent GLI-dependent oncogenic Hedgehog signaling